Key Concepts in Optical ImagingPart III: Fluorescence Techniques

Aaron Taylor, PhDManaging Director

BRCF Microscopy Core

microscopy@umich.edu

Fluorescence-Based Techniques

Why Fluorescence?

Fluorescent dyes are useful because the intensity of emissions can be quantified (unlike colorimetric stains) and sensitivity is increases since ‘background’ is dark (unlike for transmitted light).

Nucleus

Electrons

‘Energy levels’

Fluorescence

Exciting Light

-Brightness = Quantum Yield x Extinction Coefficient

-Lasts 1-10 nanoseconds-Longer wavelength-Highly Inefficient (~10-6)

GFP

FITC

Finding the Spectra for Your Dyes

Various databases show the excitation and emission spectra for virtually all fluorophores.

Review of Bright-field light path

ObjectiveLenses

Eye Piece

Eye

Perceived Image

TubeLens

Sample

CollectorLens

CondenserLens

Image Formation Visualization

Interm. Image

Lamp

Kohler Illumination

Because fluorescence is super inefficient (~10-6), the excitation light can not travel towards the objective. There is no filter good enough to separate the emissions from the much stronger excitation.

Epifluorescence Microscopy

How Epifluorescence Microscopy Works

1. Excitation light is shined through (not into) the objective.

2. The objective functions also as the condenser lens.

3. Color-selective filters distinguish the excitation from emissions.

Objective Lens ANDCondenser Lens

TubeLens

SampleInterm.ImageObj BFP

Filter ‘Cube’

Broad-band light source

Field Diaphragm

Collector Lens

Aperture Diaphragm

Field Lens

‘Condenser’ Lens

Collimated Illumination

Out of Focus Emissions are a Problem

Fluorescence generated outside the focal plane still also reaches the camera, which adds ‘blur’ that can greatly reduce image contrast and sensitivity.

ObjectiveLenses

TubeLens

ThickFluorescent

Sample Obj BFP

Filter ‘Cube’

Camera

Light from Mercury Lamp

Interm.Image Plane

Lots of out-of-focus light reaches the camera.

Typical Epifluorescence Image

Epifluorescence

Confocal Microscopy

The goal of confocal microscopy

Instead of exciting the entire sample at onve with collimated light, confocal microscopy scans a point of light back-and-forth across the sample to create an image sequentially. This design allows out-of-focus light to be blocked with a small (10’s um) aperture called a pinhole.

ObjectiveLenses

TubeLens

Excitation concentration highest in focal plane Remaining out-of-plane

emissions blocked by pin-hole

PMT

Pin-holeFocal Plane

Optical section thickness is distinct from depth of field

Optical Sectioning from a Wave Perspective

Recall that due to diffraction, light is focused to an intensity profile called an Airy disk. The width of the Airy disk is measured in ‘Airy Units’. The pinhole should be ‘just big enough’ to let most of the Airy disk pass.

PMT

Pin-hole

Intensity

Inte

nsi

ty

1D PSF2D PSF

1 AU Pin-hole

2 AU Pin-hole

Note that the Airy Units in the image plane are wavelength and magnification dependent.

How the scanning works

Mirrors attached to electric motors scan the excitation light. Feed back from the mirrors tells a computer where in the sample the point is located.

Raster Pattern

X-mirror (fast)

Y-m

irro

r (s

low

)

The scanning often determines resolution

Because the image is collected via scanning, sampling rate usually limits resolution in practice. Higher resolution can be obtained by A) decreasing the scan area or B) scanning more pixels…. as long as the point-to-point displacement remains greater than the width of the excitation PSF.

Small Scan Area; Higher Resolution

The spot size is constant and determined by the objective’s numerical aperture.

Large Scan Area; Lowe Resolution

Scan Lens

DichroicMirror

Laser Source

Scan Mirrors

A modular set of optics called the ‘scan head’ handle the point scanning, filters the excitation / emissions, and contains the pinhole and detectors.

Pinhole in Image Plane

PMT Detector

Filter

The confocal light path

Intermediate image plane

Scan head

Z-Stacks

By successively moving the axial (z) location of the focal plane within the sample, a series, or ‘stack’, of (2D) optical sections can be collected that together constitute a sampled 3D image.

ThickFluorescent

Sample

Move objective in z

A ‘3D’ Image

Other confocal designs: Spinning-disk confocal

Many points can be scanned in parallel with the use of a spinning disk of aligned lenses and pinholes. The holes are imaged onto the sample. 100s fps for one color.

Scan Pattern

Rotation of spiral hole pattern sweeps points across the scan area.

ObjectiveLenses

Camera

Fluorescent Sample

DichroicMirror

Laser Source

Focusing Disk

Pin-hole Disk

Spinning-disk confocal

A confocal optical section vs epifluorescence

Confocal Epifluorescence

When is confocal preferred over wide-field?

Epifluorescence Confocal

Resolution: -Diffraction limited - Diffraction limited (usually)

Light Path: -Wide-field -Point scanning

3D images?: -No- Sort of with deconv.

- Yes

Sensitivity: -High (single molecule) -Low (100s molecules)

Speed: - High (20 msec) - Slow (+1 sec)

Photodamage: - ‘1x’ per image - ‘10-100x’ per image

Multiphoton Microscopy

Confocal can only image about 100 um into most samples. Beyond this, too much light is lost due to absorption and scatter by the sample. Multiphoton uses pulsed infrared excitation light and specially placed detectors to reduce and work with scattered light.

100 um

Tissue

Goal of Multiphoton

T = e –Єc D

Intensity

Dep

th

NIR light penetrates tissue ~100x better than visible wavelengths

From: Weissleder. Nat Biotech. 2001

The Multiphoton Effect

Multiphoton (aka ‘two-photon’) is based on a physical process called two-photon absorption where two photons interact with an electron simultaneously to cause fluorescence. Thus, each photon needs only half the energy relative to single photon absorption.

Nucleus

Electrons

Standard Fluorescence

Exciting Light: ~490 nm

Emitted Light: ~530 nm‘Energy levels’

Multiphoton Fluorescence

Exciting Light: ~980 nmEmitted Light: ~530 nm

Scan Lens

DichroicMirror

Laser Source

Scan Mirrors

Multiphoton light path is same as for confocal, except detectors usually sit just behind objective or on sample, not behind a pinhole.

No Pinhole

Point Detector (PMT)

Filter

Multiphoton is point scanning, just like confocal

Intermediate image plane

Scan head

Multiphoton inherently optical sections

Multiphoton effect is very rare (like lighting striking twice!) so it only really happens where the excitation light is most concentrated at the focal plane. Thus, it increases as the square of the incidence power.

Focal Plane

Fluorescence only generated here.

Focused NIR excitation light

Scattered light can be used to image

Since excitation is know to happen only in the image plane, any emissions that are collected can be used for imaging. The emissions need not follow straight ray paths or even enter the objective!

Focal Plane

Scattered emissions

Scanning Electron Microscopy

SEM is also a point scanning technique

Remember electrons are charged particles. Thus, a magnetic field can be used for focusing and also for point scanning.

Ejected (SE) or scattered (BSE) electrons are detected.

Light Sheet Microscopy

Why not confocal?

Biologists often want to image large 3D volumes, but:…

1. Point scanning techniques are really slow. E.g. 10 us/px x 1M px/slice x 100 slices = 17 min per 100 slices.

2. Pont scanning techniques are harsh. High laser powers traveling through the entire sample for each image kills the sample and bleaches fluorophores.

ObjectiveLens

Fluorescent Sample

Scan MirrorsObj BFP

Point Scanning Confocal

What is light sheet microscopy?

Objective

Sample is moved or sheet is scanned to collect stack.

Camera

Excitation LightEmitted Light

Thick Sample

Light sheet microscopes use one light path for excitation and a separate light path for detection. The excitation path creates a sheet of light in the sample that is perpendicular to the detection path axis:

Many different kinds of light sheets!

All Optical LS(Scanning not required)

Scanned LSlong

Bessel Beam LS

Lattice LS

Low NA

Low NA

High NA

High NA

High NA

Cylindrical Lens

Scanned LS

Type BFP LensLongitudinalCross-Section Thickness

>2 um

>2 um

0.5 um

0.4 um

1 umshort

Different kinds of light sheets are used for different kinds samples!

Light Sheet: Summary

1) Optical sectioning is provided by plane illumination (1 um thick) and the excitation intensity is independent of imaging depth.

2) Imaging is highly parallel (wide-field) and so is very sensitive and high speed.

3) Requires an optically ‘clear’ sample to maintain the plane illumination.

4) Huge datasets (>100 GB) are difficult to analyze.

Super Resolution Microscopy

Intensity

Lens

Intensity

Recall that resolution is related to the diameter of the Airy disk, and is at best about 200 nm for a high NA oil objective.

Review of resolution

Sum Intensity

Lens

Intensity

“Resolved”

“NotResolved”

Sum

Sum

Super resolution microscopy is any technique that offers resolution better than the traditional 200 nm diffraction limit. All existing technologies are fluorescence-based.

2) Structured Illumination Microscopy (SIM)

Super Resolution Light Microscopy

1) Stochastic Optical Reconstruction Microscopy (STORM) and Photoactivated Localization Microscopy (PALM).

+ +

Fine Details in Sample

Diffraction-limited

200 nm

200 nm

200 nm

200 nm3) Stimulated Emission Depletion Microscopy (STED)

200 nm

Localization Microscopy

STORM (or PALM) ConceptUsing special dyes and lasers, it is possible to image only a few dye molecules per frame. Then, image processing can be used to estimate the location of the peak of each molecule’s image, which effectively ‘throws away’ the spread of the Airy disk. After thousands of frames, a peak localization plot (the ‘image’) can be constructed.

Peak Location

Many, many Localizations

Localization PlotObject Molecule Images

Wide-field:

Inte

nsi

ty

Image of single molecule Estimate of peak location

STORM (or PALM) is performed in TIRF mode

Wide-field Mode TIRF Mode

Glass Coverslip; RI = 1.5

Aqueous Sample; RI = 1.3

Because STORM (PALM) requires single molecule imaging, no out-of-focus light can be tolerated – the background will obscure the signal. To create an extremely thin (50 nm) optical section, these techniques are preformed in total internal reflection fluorescence (TIRF) mode.

bfp

ffp

objective

sample

excitation light

200 nm

Evanescent Field

Critical Angle

LP is certainly NOT the resolution

(5 nm pixels)

Localization density is much more limiting in practice.

E.g. with 75% of localizations recorded, resolution is around 60 nm, given 20 nm LP.

(10 nm LP)

▪ Resolution of objects is rarely better than 50 nm and usually >=75 nm.

▪ Localization microscopy is most powerful for applications where the goal is to localize individual molecules, not to resolve the size/shape of ‘objects’ comprised of many molecules.

Implications

Keep these points in mind as we talk about analysis techniques

▪ Localization of molecule positions typically 20 nm.

Particle averaging

(Laine, Nat Comm, 2015)

When all the objects are known in advance to be the same, averaging together the image of many particles can produce a higher resolution image.

Structured Illumination Microscopy

Coverslip

Sample

Two spots of excitation light are placed at a high NA position within the back-aperture (and usually a 3rd in the middle). Thus, a sinusoidal interference pattern of excitation light is produced in the front focal plane.

SIM Uses Heterodyning

objective

sample

excitation light

bfp

ffp

Moire Fringes: A 2D SIM analogy

Stimulated Emission Depletion Microscopy

STED is point scanning technique that uses an intensity annulus of light to deplete fluorescence from the edges of an Airy disk. Thus, fluorescence is generated only where depletion intensity is low, effectively narrowing the region where fluorescence occurs.

Stimulated Emission Depletion Microscopy (STED)

Airy disk of excitation light

Fluorescence generated everywhere

Annulus of depletion light superposed

Fluorescence generated only where there is no depletion

200 nm <50 nm

(Depletion emissions are removed with a filter)