Molecular Probes School of Fluorescence

Basic imaging: Chapter 1—Fundamentals of fluorescence microscopy

Simply stunningEVOS cell imaging systems

Compare systems at thermofisher.com/simplystunning

For Research Use Only. Not for use in diagnostic procedures. © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. CO016293 0816

Minimize the complexities of microscopy without compromising performance. Smarter systems | Easier cell imaging | Faster results

cell imaging

1

Physical properties that define fluorescence ..........2The visible spectrum of light .........................................2Range of detection in fluorescence microscopy ...........2What is fluorescence? ..................................................3Where the fluorescent signal comes from .....................3Excitation and emission spectra ...................................4The all-important Stokes shift .......................................4

How fluorescence microscopy works ......................5Microscopy is a basic research tool ..............................5What is a microscope? .................................................5Brightfield vs. fluorescence microscopy........................5The difference between magnification and resolution ...6Using fluorescence can increase resolution ..................6

Epifluorescence microscope basics ........................7Light and resolution in epifluorescence microscopes ...7Epifluorescence microscopes .......................................7The light path in epifluorescence microscopes .............8Magnification vs. resolution ..........................................8Limits of resolution in epifluorescence microscopy .......9Upright vs. inverted microscopes .............................. 10

Contents

Chapter 1—Fundamentals of fluorescence microscopyIn this chapter you will learn the basic physics behind the excitation and emission of a fluorophore and how microscopes channel and capture the light generated to make a fluorescence-based image.

Access this content at thermofisher.com/mpsf-imagingfundamentals

Using filters to capture your signal ......................... 11Filters help you control light during imaging ................ 11Why fluorescence microscopy uses filters .................. 11The different kinds of filters used in epifluorescence imaging ............................................ 11

Understanding your objective ................................. 12Match your objective to your imaging task .................. 12Understanding your objective ..................................... 12Selecting the right magnification ................................. 13Matching immersion medium to objective................... 13Refractive index .......................................................... 14Numerical aperture and the refractive index of immersion medium ....................................... 14Objective type ............................................................ 15Coverglass thickness ................................................. 15Correction collars ....................................................... 15Working distance ....................................................... 15

Additional resources ............................................... 16Glossary ................................................................... 18

2

Physical properties that define fluorescenceFluorescence is a function of light energyFluorescent molecules by definition absorb light at one color (wavelength) and emit it at another. The difference in colors is called the Stokes shift. The cameras used in fluorescence microscopy allow the detection of signal beyond the wavelengths our eyes can see. In this section you will learn about the physical properties that define fluorescence, including wavelength, how energy relates to fluorescence and fluorescent colors, and what defines a fluorescent molecule’s spectra.

The visible spectrum of light When we talk about light in microscopy, it is usually noted as wavelength, even though photons (the packets of energy that make light) can act as both particles and waves (Figure 1.1). Visible light, or light that we can see with our eyes, is usually in the range of 400–700 nm and encompasses all colors in the rainbow, with blues starting at around 400 nm and reds finishing at around 700 nm.

Range of detection in fluorescence microscopyThe range for fluorescence imaging extends a bit beyond where our eyes can see. Using this extended range is no problem because the CCD cameras that collect light emitted from our sample in a typical fluorescence microscopy setup have a wider range than our eyes do (Figure 1.2). In practical terms, imaging wavelengths for cell biology are usually in the range of 300–800 nm.

Figure 1.1. The electromagnetic spectrum, with visible wavelengths and their corresponding colors highlighted.

Figure 1.2. The range and efficiency of light detection for a CCD camera compared to that of the human eye.

3

Figure 1.3. The inverse relationship between energy and wavelength in the visible spectrum.

Figure 1.4. Simplified Jablonski diagram showing the energy state change of a fluorophore’s electron as it undergoes fluorescence, with the corresponding change in the color of light.

What is fluorescence?But what is special about the term fluorescence? Fluorescence refers to the physical property of an object absorbing light at one wavelength and then reemitting it at another wavelength. If a molecule absorbs the light of one wavelength and emits it at another (i.e., fluoresces), we call that molecule a fluorophore. Usually the wavelength the molecule emits will be of lower energy than what it absorbed (Figure 1.3), so in simpler terms we could say that something absorbs blue light and emits green, or absorbs green light and emits red.

Where the fluorescent signal comes fromTo understand this at a deeper level, we need to think about photons, the packets of energy that make up light. The magnitude of energy that a photon contains determines its color or, in physical terms, its wavelength. When the light (or photon) hits a fluorophore, the energy is transferred to the fluorophore’s electrons. The electrons are excited, but then rapidly lose that extra energy (that sounds like people too, doesn’t it?). The end result of this loss of energy is the emission of a photon of light, but that photon will have less energy than the original photon, so it will have a longer wavelength and be a different color (Figure 1.4). The emitted photons are the signal you need to collect as data during your imaging experiment.

4

Excitation and emission spectraMost fluorophores don’t just absorb light at one discrete wavelength and emit light at another discrete wavelength: they usually absorb and emit a range of wavelengths. So when we think about using fluorophores in imaging, it’s useful to also think about the entire spectrum of their absorption and emission, while at the same time keeping in mind the maximum excitation and emission wavelengths (Figure 1.5). The maximum values are the peaks of the excitation and emission spectra.

The all-important Stokes shiftThe difference between the excitation and emission maxima for a given fluorophore is called the Stokes shift. A fluorophore with a large Stokes shift will be much easier to use in your imaging than a fluorophore with a small Stokes shift. When there is only a small difference in wavelength between excitation and emission, it will be very difficult for you to see the emitted light from your labeled object as separate from the light used for excitation, and there will be more problems with background fluorescence.

Figure 1.5. Excitation and emission spectra of a nuclear dye (DAPI). Shows both the fraction of light absorbed by the dye over a range of wavelengths (excitation, shown in purple) and the light emitted from the dye over a range of wavelengths (emission, shown in blue).

Figure 1.6. A fluorophore with good separation between the excitation and emission maxima typically results in more reliable detection than a fluorophore with little separation. Compare the fluorophore with a large Stokes shift (purple and blue maximum peaks) to a fluorophore with a small Stokes shift (orange and red peaks).

5

Figure 1.7. A magnifying glass is the simplest form of lens used to view objects.

Figure 1.8. (A) The light path through lenses and sample in basic brightfield microscopy, and (B) antique 19th century drum-style compound microscope.

Microscopy is a basic research toolMicroscopes are powerful research and discovery tools and have contributed to countless groundbreaking discoveries, over several centuries. In this section you will find basic definitions for common microscopy terms, information to help you understand differences between magnification and resolution, and how fluorescence can be used to improve contrast and increase resolution.

What is a microscope?A microscope magnifies objects; more lenses translate to higher magnification. In its most basic form, a microscope is simply a device that allows you to see things that are not visible to the naked eye, by the use of lenses to magnify your sample (Figure 1.7). That means a magnifying glass is technically also a microscope. If you use light along with a lens, you have something called an optical or light microscope.

Brightfield vs. fluorescence microscopyIf you use additional lenses to increase your ability to magnify a sample, you have a compound microscope (Figure 1.8). Most modern microscopes are compound microscopes, because the additional magnification gives a more enlarged image. If only white light is used for illumination, then it’s brightfield microscopy.

How fluorescence microscopy works

6

The difference between magnification and resolutionIt’s pretty important to understand the difference between magnification and resolution when it comes to getting a good result when you’re doing fluorescence imaging. When we talk about magnification, we are referring to how much bigger an object appears when we look at it under the microscope.

In contrast, when we talk about resolution in a practical sense, we are referring to how much detail we can distinguish in our image, which can be subjective. In a more technical sense, resolution is limited by the refractive properties of light.

Using fluorescence can increase resolutionResolution and magnification are not the same. In microscopy, we refer to ability to distinguish details in a magnified image as resolution. Magnification, or making something appear larger, doesn’t do you much good if you cannot see the detail in the sample you’ve magnified.

Brightfield microscopy relies on the differences in absorption of light due to differences in densities between various parts of the sample, which for our purposes is a cell. Because of this, brightfield microscopy is not great when you want to see many details in your cell. How do you increase your resolution? Add in fluorophores to stain structures in your sample and filters to illuminate it, focus the light emitted by the sample, add a sensitive detector, and voila! Now you can do fluorescence microscopy. Fluorescence microscopy gives you the advantage of better resolution by making various structures in the cells contrast better with their neighbors, as well as allowing you to collect images in more than one color (Figure 1.9).

Figure 1.9. An image of the same field of BPAE cells captured using brightfield (top) and fluorescence (bottom) microscopy. Fluorescent labeling of the nucleus (yellow) and actin (red) makes it possible to see much more detailed cell structure.

7

Figure 1.10. A dichroic beamsplitter allows longer wavelengths of light to pass through the filter while reflecting shorter wavelengths of light.

Light and resolution in epifluorescence microscopesBy placing your sample on the stage and directing light through it, the filters, detectors, and camera, you will be able to visualize and capture an image of your sample. In this section you will learn about basic light path and filter configurations, what governs the limit of resolution, and the differences between upright and inverted scopes.

Epifluorescence microscopesYou really don’t need to know in great detail about how a microscope works in order to do fluorescence imaging, but it can help a little when it comes to troubleshooting which, really, given how iterative science is, ends up being about 99.8% of the time, right? And one of the things that you probably do need to understand if you’re trying to solve an imaging problem is the filter setup for your fluorophore.

Epifluorescence microscope basics

Most fluorescence imaging is done using fluorescence microscopes that have these essential components (Figure 1.10):

• A light source: usually a xenon arc or mercury vapor lamp, but more recently, powerful LEDs

• A filter (for incoming light): narrows the wavelengths of the incoming light to only those used to excite the sample; it is called, the excitation filter

• A dichroic beamsplitter or mirror: to reflect the excitation light to the sample and simultaneously transmit only the emitted light from the sample back to the detector (Figure 1.10)

• A filter (for emitted light): transmits only the wavelengths of the emitted light from the sample and blocks all the light passed through the excitation filter; it is called, the emission filter

• A CCD camera: Emitted light does you no good if you can’t detect it, and for fluorescence imaging the detector is usually a CCD camera. The camera is usually also connected to a computer screen, which shows you an image of your sample.

8

The light path in epifluorescence microscopesFigure 1.11 shows the typical light path of an epifluorescence microscope. Most microscopes that are used for cell biology are arranged so that the light travels through the objective lens to illuminate the sample, and then the light emitted from the sample travels back through the same objective to the detector.

This arrangement—where both the illuminated and emitted light travel through the same objective lens—is referred to as epifluorescence microscopy, where “epi” is borrowed from the Greek to mean “same”. A more correct term would be epifluorescence illumination, but most people assume the illumination part, since fluorescence depends on illumination. A transillumination fluorescence microscope is not as common, but you still may encounter a setup where the illumination and collection of signal are on opposite (trans) sides of the stage with the sample in between (Figure 1.12).

Magnification vs. resolutionMagnification It’s pretty important to understand the difference between magnification and resolution when it comes to getting a good result when you’re doing fluorescence imaging. When we talk about magnification, we are referring to how much bigger an object appears when we look at it under the microscope (Figure 1.13).

Resolution In contrast, when we talk about resolution in a practical sense, we are referring to how much detail we can distinguish in our image, which can be subjective (Figure 1.14). In a more technical sense, resolution is limited by the refractive properties of light.

Figure 1.12. Inverted and upright microscopes both utilize epifluorescent illumination: the main difference is the location of the objectives relative to the stage where the sample is placed.

Figure 1.11. Typical light path in an epifluorescence microscope. Notice that both the excitation and emission are controlled by the dichroic mirror, which reflects excitation light (shorter wavelengths) onto the sample and passes the resulting emission light (longer wavelengths) through the filter and on to the detector (the viewer or the camera).

9

Figure 1.14. Same images matched in size to show differences in resolution.

Figure 1.13. Two 6 μm beads taken at 3 different magnifications: 4x, 10x, and 40x.

Figure 1.15. The resolving power of various microscopes, with representative objects within range for both light microscopes and electron microscopes.

Limits of resolution in epifluorescence microscopyWhat does this actually mean? It means that a typical epifluorescence illumination compound microscope cannot resolve or distinguish two objects that are less than 200 nm apart. Additionally, because the whole sample is illuminated at the same time, you are detecting all of the in-focus and out-of-focus light in your sample. These limitations mean that, depending on the lenses in your objectives, you will be able to determine that two differently colored probes are present in the same cell, but you may not always be able to resolve their spatial relationship to each other without a lot of controls, individual pixel analysis, and math. Also, because you don’t have any information about depth, you can’t really draw any sound conclusions about volumes from an image taken with an epifluorescence microscope. By understanding and working within the limitations of your system (Figure 1.15), you can be confident in the data and images you collect, as well as being able to fully understand your data and formulate conclusions.

10

Upright vs. inverted microscopesYou will sometimes hear people refer to microscopes as upright or inverted. These terms refer to the location of some components, like objectives and light sources. Upright microscopes have objectives placed above the stage where you put your sample; inverted microscopes have objectives below the stage where you put your sample (Figure 1.12).

There’s no fundamental difference in the ability of upright and inverted microscopes to produce and channel light along various paths (Figure 1.16). The image quality you are able to achieve will have more to do with your sample preparation, lenses, light source and wavelength, fluorophore filter set, and camera than the locations of components on the microscope. Some experiments will require a particular orientation in order to accomplish what you need, so it’s always a good idea to look at a new-to-you microscope and walk yourself physically through the steps of your experiment to make sure the setup will work for you.

Figure 1.16. Epifluorescence microscope light paths. The yellow line represents the arrangement of the light path for brightfield illumination. The illumination light does not travel through the objective, only the transmitted light from the sample. The blue line illustrates the path of excitation light, which travels through the filter cube and objective to the sample, and the resultant emission light (shown in green) simultaneously travels through the objective and filter cube and the detectors. In epifluorescence microscopy, both the excitation and emission light travel through the same objective.

Laser scanning confocal microscopy still relies on a compound light microscope setup but can give you more resolution. The increase in resolution comes from the use of lasers for illumination, which narrows the excitation range to ~2–3 nm. This is around 10 times narrower than the range of wavelengths you get when using excitation filters. Additionally, the ability to obtain an image from just one focal plane—while removing all of the scattered and out-of-focus light generated in an experiment—can also increase resolution. The restriction to one focal plane is accomplished using a pinhole to block out-of-focus light before it gets to the detector; this is referred to as optical sectioning. The pinhole permits light from only a very narrow section of the sample and gives you information about depth. This is an improvement over the resolution you can get using epifluorescence, which collects the light from many focal planes within a cell. There are other alternatives for scientists who want more resolution, but they tend to be more specialized and require greater technical knowledge to get started.

11

Figure 1.17. Excitation and emission spectra of a nuclear dye (DAPI) overlaid with the range of wavelengths passed through the filters for excitation (purple shading) and emission (blue shading). The black line depicts the transmission of the dichroic filter.

Filters help you control light during imagingBy placing filters in various spots in your epifluorescence microscope, you can define the wavelengths of light that excite your sample and the wavelengths that you detect in the fluorescence emission. In this section you will learn about various filters used in epifluorescence imaging.

Why fluorescence microscopy uses filtersFor epifluorescence microscopes, the excitation filter, dichroic beamsplitter, and emission filter will usually all be housed in the same cube and will be designed to match the spectra of a specific fluorophore or fluorescent protein. Occasionally the filters will not be in a cube, but will be on a wheel, referred to as a turret. Turrets allow you to mix and match your excitation and emission wavelengths. The ultimate goal is to collect the light you want from the fluorophore bound to your target and not any light from other parts of the system (ambient light, excitation source, etc.). We refer to unwanted light that makes it to the detector as background fluorescence.

Using filters to capture your signal

The different kinds of filters used in epifluorescence imaging In general, a filter set will be designed to capture the maximum excitation and emission wavelengths of a given fluorophore, but will not capture all of the fluorescence (Figure 1.17). Most modern filter sets are designed so that the excitation filter has a defined band of wavelengths that it allows through. This style of filter is referred to as a bandpass filter. Bandpass filters are normally identified by the middle-value wavelength and the width of the band. In a simple epifluorescence microscope setup, once the excitation light leaves the excitation filter it is reflected onto the target. The fluorophores in the target become excited and then emit light that is shifted towards the red end of the spectrum (compared to the excitation light). Not all of this emitted light will be captured by the detector—only what is allowed through the dichroic beamsplitter and also passes through the emission filter. Emission filters are usually bandpass or longpass filters, depending on the specific fluorophore and imaging experiment. A longpass filter may be desirable if you need all the light beyond a certain wavelength to pass through to the detector.

12

Understanding your objectiveMatch your objective to your imaging taskA microscope objective is composed of a complex set of lenses and optics, and different objectives are designed for different imaging tasks. Capturing good images relies on choosing the correct objective. In this section you will learn the correct magnification for your experiment and how to tell if your objective can be used with air, oil, or other immersion media.

Understanding your objective The objective is an essential part of the microscope and can greatly influence image quality. Objectives come with lots of information written on them, and most of it is written in code. But don’t worry; it’s easy to decipher. Common notations found on objectives are indicated in Figure 1.18. Read on for more detailed descriptions of each.

Figure 1.18. Common notations found on objectives.

13

Selecting the right magnificationMagnification tells you the optical magnification the objective provides. The magnification you choose depends on what you want to see. The usefulness of magnification will be limited by your resolution. Making a big fuzzy blob of light even bigger won’t give you a better picture (Figure 1.19). Provided you have similar resolution at different magnifications, using higher magnification will allow you to see smaller things (such as organelles inside a cell) better. On the other hand, using a lower magnification will give you a better image of the big picture—such as a field of cells or interactions between cells.

Matching immersion medium to objectiveThe immersion medium is what’s between the objective and the coverslip (or the bottom of the dish or flask that holds your sample). Each objective is designed for a specific immersion medium, which is marked on the objective (Figure 1.20). The main types of immersion media are air, oil, and water. It is important that you never put air objectives in oil or other liquids. Doing this will make the person in charge of the microscope really angry! The main purpose of using different types of immersion media is to minimize the refractive index differences that are present in the space between the objective and the sample. This includes the substrate (i.e., glass coverslip) that the sample is on and the imaging medium (i.e., buffer) that the sample is in. Minimizing the differences will result in better image resolution.

Figure 1.19. Same field of cells captured at different magnifications. Each magnification can offer different information, and the best choice for your experiment will vary depending on what you want to know.

Figure 1.20. Use of immersion media matched to the objective can minimize the refractive index differences between the objective and the sample.

14

Refractive indexLight will travel through different types of materials at different rates. When light travels through one material (such as air) and into another (such as water), the light is refracted. It appears bent (Figure 1.21). For instance, when you put a pencil in a glass of water and view the glass from the side, the pencil will look bent. This is because air has a refractive index different from that of water.

Numerical aperture and the refractive index of immersion mediumNumerical aperture is a property of the objective that indicates how good the resolution can be in the image you collect (basically how much fine detail you can see). Often, you will hear people talk about the “NA” of an objective. “NA” stands for numerical aperture, and its value partly depends on the refractive index of the material that is between the objective and the glass coverslip that your sample is on. In general, objectives with higher NA give you better resolution. Higher-NA objectives often have higher magnification and are used with some sort of immersion medium. Immersion media are used to alter the refractive index of the space between the objective and glass coverslip so that it is closer to the refractive index of the glass coverslip itself. This minimizes refraction and loss of light, ultimately giving you a better image.

Figure 1.21. The pencil appears bent or broken because the refractive indexes of water and glass are different than from of air.

15

Other information on your objective

Objective typeThe objective type indicates how many corrective lenses the objective contains for various types of aberrations which can help with resolution. Corrective lenses can help correct things like the way the light is bent as it goes through the objective so that the edges of your field are as crisp as the middle, or they may correct for the fact that different wavelengths of light will behave differently as they pass through the lenses in the objective.

Coverglass thickness Samples are often mounted on glass coverslips. The most common glass coverslip is named #1.5 and has a 0.17 mm thickness. The number for the coverglass thickness that is printed on the objective tells you the optimal thickness of glass coverslip (or any other type of substrate your sample is on, such as a plastic-bottom dish) the objective is manufactured for.

Some objectives have a long working distance, meaning that the objective can give you a good image over a large range of coverglass thicknesses. This is useful if you want to image through thicker vessels such as plastic-bottom dishes and T-75 flasks, which have thicker walls than glass- bottom vessels. To give the best imaging results, the lenses in the long working distance objective are manufactured to accommodate differences in coverglass thicknesses.

Correction collarsSome objectives have a manual correction collar. You can turn the correction collar to a specified number that indicates the thickness of the coverslip your sample is on. Not all long working distance objectives have correction collars, though.

Working distance The working distance is the distance between the objective and the cover glass, or between the objective and the top (or bottom) of whatever vessel you are imaging through, when your sample is in focus. When you are imaging through something thin, like a coverglass, you can use objectives with shorter working distances. But when you are imaging samples that are in thicker vessels, such as plastic plates or dishes, you will probably need an objective that has a longer working distance. The working distance of an objective is often written on the objective. The working distance of the second objective in Figure 1.18 is 7.4 mm. It is considered to have an “extra-long working distance” and is abbreviated as ELWD on the objective.

16

Additional resources for fundamentals of fluorescence microscopyTutorial videos on the basics of fluorescenceIntroduction to fluorescence: This video provides an easy-to-understand overview of the basic principles of fluorescence and is suitable for beginners or for those that need a quick refresher.

Anatomy of fluorescence spectra: This video describes the principles behind fluorescence spectra and how they can be used to determine properties of a fluorescent molecule.

Overview of filters and light sources: This video describes how fluorescence filters work and the various light sources used for fluorescence excitation.

Useful web linksMolecular Probes School of Fluorescence thermofisher.com/mpsf

Additional chapters for imaging basics:

Chapter 2—Sample considerations

Chapter 3—Labeling your samples

Chapter 4—Capturing and analyzing your samples

Chapter 5—Protocols and troubleshooting

17

Useful web tools to help select dyes and stainsFluorescence SpectraViewer: Plot and compare spectra and check the spectral compatibility of multiple fluorophores.

Online cell stain tool: This tool will help you select the right combination of fluorescent stains and dyes by providing a visual image of the products in use. Create your own cell image and know what your cells will look like before you buy the products.

Fluorophore selection guide: This guide offers a quick view of the most commonly used fluorophores and their physical properties to help you select the right one for the right application and instrument setup.

18

Glossary

A

Antigen A material that causes an organism to have an immune response and generate antibodies.

B

Background fluorescence Nonspecific and unwanted fluorescence produced by a sample, vessel, or imaging medium, or from fluorophores not bound to specific targets.

Bandpass filter An optical filter through which only light of a given wavelength range can pass. All light of higher or lower wavelengths is blocked. Normally identified by the middle-value wavelength and the width of the band.

Binning Combining a group of pixels into a single pixel to reduce the total number of pixels in an image. This can reduce image noise and shorten the necessary exposures times but can also lower resolution.

Blebbing Formation of protrusions from a cell membrane; these eventually separate from a cell during cell death.

Bleed-through The incorrect detection of emitted light from a fluorophore in a channel intended for another fluorophore.

Blocking solution A solution used to prevent nonspecific, low-affinity binding in ICC samples.

Brightfield microscope A light microscope in which the sample is illuminated with white light and images are formed due to the light-absorbing properties of structures within a sample.

C

CCD camera Charge-coupled device; an image sensor that converts light into electrons to create digital images.

Cell-permeant Able to freely diffuse across the plasma membrane, into and out of a cell.

Cellular membrane Phospholipid bilayer that forms the outer barrier of a cell or encloses various compartments within a cell.

Crosslinking A bond that links proteins with other nearby nitrogen atoms in protein or DNA through a –CH2– linkage.

Cytoskeleton The network of filaments and tubules that provides a cell’s structure and shape.

D

Detector The part of a fluorescence imaging system that detects the signal from a sample. This could be the oculars on the microscope directing light to your eyes, or an electrical component such as a camera that records photons and creates digital images.

Dichroic beamsplitter (filter)

An optical device that splits a beam of light, reflecting the shorter wavelengths (a defined range) and allowing all other higher wavelengths to pass through.

Dynamic range The range of light intensities detectable by a camera, those that are between background noise and pixel saturation.

19

E

Emission Photons emitted from an object as excited fluorescent molecules relax to a lower-energy state.

Emission filter An optical device that limits the emitted light collected from a sample to a defined set of wavelengths.

Epifluorescence When illumination light and emission light travel through the same objective lens.

Epifluorescence illumination

The most common arrangement of optical components in a fluorescence microscope, in which excitation and emitted light both pass through the same objective lens, which is located above the sample.

Epithelial A type of animal cell that lines internal and external surfaces of the body.

Epitope The specific area on a target molecule that an antibody binds to.

Excitation filter An optical device that limits the excitation light applied to a sample to a defined wavelength range.

Excite To drive an electron in a fluorophore to a higher energy state as a photon of light is absorbed.

Exposure time The length of time a camera’s detector is exposed to light emitted from a sample while capturing an image.

Extinction coefficient A value that expresses the capacity of a fluorophore to absorb light at a given wavelength in vitro.

F

Fibroblast A type of animal cell typically found in connective tissue.

Fluorescence The emission of light from a sample that occurs as excited fluorescent molecules relax to a lower energy state.

Fluorescence microscope A light microscope that creates images based on the emission of fluorescent light from a sample.

Fluorescent dye Also called fluorochrome; a fluorescent chemical compound that can re-emit light following light excitation.

Fluorescent proteins A class of proteins that contain amino acid sections that can act as fluorophores.

Fluorophore A fluorescent chemical compound that can re-emit light following light excitation.

Fluorophore filter set A selection of excitation, emission, and dichroic filters designed for detecting light from a given fluorophore.

Fusion protein A protein formed by the expression of an artificial gene formed by linking two or more different genes or gene fragments.

20

G

Grayscale Composed of shades of gray ranging from white to black.

H

HBSS Hanks’ Balanced Salt Solution; a saline-based solution that has a physiological pH and salt concentration.

Heat-inactivated serum Serum that has been heated to 56°C for 30–60 minutes to remove enzymatic or other protein activity normally present.

I

Immunocytochemistry Abbreviated as ICC. In this technique, a primary antibody is used to localize a specific protein (target) in a cell. The primary antibody specifically binds to the target and is usually visualized microscopically by binding of a secondary antibody that has a fluorescent label.

Immunofluorescence The use of antibodies labeled with fluorescent dyes to detect a specific protein in a cell.

Immunofluorescent labeling

A technique that uses fluorescently labeled antibodies to detect and visualize specific protein biomarkers in a cell.

Immunolabeling intensity In the context of immunofluorescent labeling, a measure of the quantity of light per given area of sample.

Inverted microscope A microscope with objectives placed below the sample stage; samples viewed on this type of microscope will have to be inverted on the stage.

L

Laser scanning confocal microscope

A compound light microscope that uses lasers as excitation sources and pinholes to limit the detected light to one focal plane.

Light The range of electromagnetic radiation visible to the human eye.

Light path The sequence of mirrors, lenses, and filters through which light passes from the excitation source to the detector.

Light source A device such as a lamp or laser used to illuminate a sample.

Lipid transfection The use of lipid micelles to insert genetic (DNA or RNA) into mammalian cells.

Long working distance Description of a type of objective lens that can provide a good image with a range of coverglass thicknesses.

Longpass filter An optical filter through which only light above a given wavelength range can pass. All light with lower wavelengths is blocked.

21

M

Magnification The visual enlargement of a sample, viewed with a microscope.

Membrane potential The difference in electrical potential across a cellular membrane such as the plasma membrane or mitochondrial membrane.

Metabolism The collection of biochemical reactions occurring in a cell.

Mitochondria Organelles in eukaryotic cells whose function is to generate ATP.

Mounting medium Any medium in which the sample is bathed during imaging.

N

Noise Nonspecific and undesired fluorescence produced by components of a microscope, including the excitation source, camera, and external light source.

Nonspecific binding Noncovalent binding that is not specific to a particular target, ligand, or epitope. It is thought to be due to “stickiness”, or hydrophobic interactions, and can be minimized with additional washes and/or by adding competing hydrophobic molecules.

Numerical aperture A measurement of the ability of an objective to gather light and provide a high-resolution image.

O

Osmolarity Also referred to as osmotic concentration; it is the number of osmoles of solute per liter of solution (Osm/L).

P

PBS Phosphate-buffered saline. A solution containing water, sodium chloride, sodium phosphate, and in some cases, potassium chloride, and potassium phosphate. The ions and osmolarity are matched to those found in the human body.

Photobleach curve An exponential decay curve that displays the reduction in fluorescence as a fluorophore is continuously exposed to light.

Photobleaching Destruction of an excited fluorophore due to photosensitized generation of reactive oxygen species.

Photon The smallest discrete unit of light.

Photostability The ability of a fluorophore to resist signal degradation from photobleaching.

Phototoxicity Damaging effects on live cells caused by light or by the generation of reactive oxygen species that follows the illumination of a fluorophore.

Pixel The smallest discrete area of an image.

Plasma membrane The membrane surrounding an animal cell which is composed of a phospholipid bilayer and proteins.

Primary antibody An antibody generated to specifically recognize (bind to) a target of interest (targets can be proteins, peptides, carbohydrates, phosphorylation sites on proteins, or other small molecules). A good primary antibody will have both high affinity (binds tightly to the target) and specificity (doesn’t bind tightly to any other molecule).

Pseudocolor To apply color to a given pixel in an image; usually the color will match the emission color of the fluorophore.

22

Q

Quantum yield A measure of efficiency of a fluorophore, defined as the fraction of absorbed photons that result in emitted fluorescence in vitro.

H

Refractive index A measure of how much light is bent as it passes through two objects of different composition.

Resolution The shortest distance between two points on a sample that can still be distinguished as separate.

Ringer’s solution A clear buffered salt solution that is isotonic and has a physiological pH.

S

Sample The object to be imaged; typically a biological sample such as tissue or cells.

Saturated A condition when a given pixel in a camera has collected the maximum number of photons. Additional detected photons will not be counted beyond the saturation point.

Secondary antibody An antibody generated to recognize (bind to) other antibodies, typically labeled with a probe that makes the complex visible under microscopy. Noncovalent binding involving hydrogen bonds, van der Waals forces, and ionic bonds mediate the specificity between an antibody and a target or epitope.

Specific binding Specifically bound molecules cannot be easily washed away with PBS or blocking buffer.

Stokes shift The difference in wavelength between the excitation and emission maxima of a fluorophore.

Subcellular Smaller than, and generally contained within, a cell.

T

Transduction The process of inserting genetic material into a cell using a viral vector.

Transfected Cells that contain inserted genetic material (DNA or RNA).

U

Upright microscope A microscope with objectives placed above the sample stage; samples viewed on this type of microscope will have to be placed upright on the stage.

V

v/v Volume percent concentration, v/v = [(solute volume)/(solution volume)] x 100%. For example, a 5% v/v solution would contain 5 mL of solute diluted to a final volume of 100 mL.

Viability assay One or more fluorescent reagents used to determine the health of cells in a sample.

W

w/v Weight/volume percent, w/v = [(solute weight, in g)/(solvent volume, in mL)] x 100%. For example a 5% w/v solution would contain 5 g of solute in 100 mL of solvent.

Washing A rinse after incubations to dilute or remove the non specifically bound original material, generally performed using PBS.

Wavelength The distance between peaks of two consecutive light waves.

Quantifiably brilliant data

High-content analysis is now open to allAffordable form and function come together in the simple yet powerful Thermo Scientific™ CellInsight™ CX7 Imaging System—a brilliant integration of seven-channel confocal detection, four-channel brightfield imaging, Invitrogen™ reagents, image analytics, and database tools. Now, your lab can afford to get rich quantitative data.

Driven by Thermo Scientific™ technology. Powered by Invitrogen reagents.

Find out more at thermofisher.com/cx7

For Research Use Only. Not for use in diagnostic procedures. © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. CO125356 0816

Find out more at thermofisher.com/molecularprobes

For Research Use Only. Not for use in diagnostic procedures. © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. COL01959 0816

The images and graphics in this publication are copyrighted, but they are freely available for your use as long as the attribution “Molecular Probes School of Fluorescence” remains intact.