MULTI-PHOTON MICROSCOPY: APPLICATIONS AND THEORY … and MP… · multi-photon microscopy:...

MULTI-PHOTON MICROSCOPY:APPLICATIONS AND THEORY

PART I

JAMES LOPEZ PH.D.

NATIONAL SALES APPLICATIONS SPECIALIST

FLUOVIEW LASER SCANNING CONFOCALAND MULTIPHOTON

Microscope Optics - Primer for microscopy

3 KEY PARTS OF THEMICROSCOPE SYSTEM

1)OBJECTIVE

2)FILTERS

3)DETECTION

Microscope Optics - Primer for microscopy

Lateral and axial chromatic aberration

Spherical aberration

smb://upload.wikimedia.org/wikipedia/commons/1/10/Spherical-aberration-slice.jpg

Refractive Index

refractive index =

Speed of light in a vacuumSpeed of light in medium

Air n = 1.00

Water n = 1.33

Silicone Oil n = 1.40

Glycerol n = 1.47

Immersion n = 1.52Oil

TIRF n = 1.78Oil

Numerical Aperature Numerical aperature (NA) =

n (sin (µ))

Theoretical maximum for µ = 90º

n = refractive index

40x UPlanSApo 0.95NA Dry

n = 1.00 AIR

1.00 (sin (90)) = 1.00 NATheoretical maximum

40x UPlanSApo 0.95NA Dry

40x Dry UPlanSApoN 0.95NA is a great objective,but how does this compare to the 40x Oil UPlanFlN 1.3NA?

R=λ/(2NA)R=(0.61λ)/NA

Resolution is NA dependent

Resolution is the ability to separate two objects

40x Dry UPlanSApo 0.95NAR = 500nm/(2 x 0.95NA)R = 263nm

40x Oil UPlanFl 1.3NAR = 500nm/(2 x 1.30NA)R = 192nm

Oil vs. Dry Increase NA by:1)Increasing ½ angle aperture (µ)

(by reducing WD)

2) Increasing n (refractive index) (by using oil)

NA = n (sin (µ))

Numerical Aperature

High NAShallow depth of field (short focal distance low F stop)(Thin optical slice)Shorter working distance( Light comes to a focus close to the

objective face)

Low NADeep depth of field(Thick optical slice)Long working distance( Light comes to a focus far from the objective face)

Why would someone want a low NA lens?

NA in image brightness

Fluorescence - light starved application.Maximize that signal.

ImageIntensity

= NA^4Magnification^2

For fluorescence

40x Dry UPlanSApoN 0.95NA40x Oil UPlanFlN 1.3NA

0.95^440^2 = 5.09 1.30^4

40^2 = 17.85

Refractive index matching

Specimen n = ~1.38

Silicone oil n = 1.40Water n = 1.33Glycerol (0.75% in water) n = 1.44Immesion Oil n = 1.52

40x UPlanSApo Silicone Oil Objective

NA - 1.25WD 300 micronsRefractive index matchingChromatically correctedSpherical aberration correction collar

Objective Transmission

60x Oil 1.35 UPlanSApo 60x Oil 1.42 PlanApo

FLUORESCENT MINERALS

UV excitation/absorbanceHigh energy short wavelengths

Red-shifted emission Lower energy long wavelengths

FLUORESCENCE OF GFP

GFP 1Pabsorbance

GFP emission

λ

λ

λ

GFP:Blue excitationGreen emission

%

%

%

An advanced view on fluorescence

WIDE-FIELD MICROSCOPY

WIDE-FIELD CONFOCAL

ASPECTS OF CONFOCAL MICROSCOPY

DILEMMA OF MICROSCOPY - ACTUAL CELL IS 3-D

Spinning DiskConfocal

SectionDepth

Background

Background

Wide-field Epi-Fluorescence

LaserScanningConfocal TIRF

Fluorescence Imaging Modalities: Optical Sectioning

LASER SCANNING CONFOCAL FOR OPTICAL SECTIONING

PINHOLE 800µm MAXIMUM INTENSITY PROJECTION

PINHOLE 80µm MAXIMUM INTENSITY PROJECTION

Epi-Fluorescence

WIDE-FIELD FLUORESCENCE IMAGE OF LOBELIA

Disk Confocal

SPINNING DISK CONFOCAL IMAGE OF LOBELIA

Phase Phase

DIC Nomarski DIC Nomarski

DIC Nomarski microscopy

Pros: Full NA (no phase ring), optical sectioning, no haloCons: expensive, more difficult of a setup compared to phase

Polarize lightSheer lightFocus lightDifferences in refractive indexCombine lightAnalyzer (reject direct light)

Fang-Yen Lab

Olympus FV1000MPE Multi-Photon Imaging System

FluoView FV1000 Overview Visible Confocal Features

Main Scanner ： FL3CH ＋ Transmitted detectorSIM Scanner: A second independent, but fully

synchronized laser scanner incorporated in a single compact design for simultaneous laser stimulation and observation.

Lasers: Multi-line Ar （ 458, 488, 515nm ） HeNe （ 543 ）

Laser Diode (405, 440, 473, 559, 635) AOTF: standard equippedTwo types of Detectors: Filter, SpectralApplicable Microscopes: IX83 ， BX61 ， BX61WI

FluoView FV1000 Scan Head Overview

Modular FV1000 Scanning Head

Detector Box(Spectral and/or filter)

Main ScannerBox

SIM ScannerBox

4th Detector orFiber Output


FV1000 Components Fluorescence Turret BX61WI

Fluorescence turret has factory aligned mirror at position one.

Adaptor for BX61WI


FV1000 Components Fiber Port - Fiber coupling output

• compatible fiber core 100-125μm

Fiber fluorescence light output for FLIM or spectrophotometer, etc.

FluoView FV1000 Overview

SIM Scanner Applications Photobleaching

FLIP fluorescence loss in photobleaching

FRAP fluorescence recovery after photobleaching

FRET by acceptor photobleaching Photoconversion Photoactivation Uncaging


SIM Scanner

Stimulation or Photobleaching Using SIM scanner with 405nm LD

Confocal Imaging473nm ＆ 559nm

Laser stimulation or photobleaching synchronized with confocal fluorescence imaging - ChR


SIM Scanner Tornado scan for rapid photobleaching or activation

Tornado Scan Conventional Scan

Laser on and offNon stop illumination!

Fast results with no recovery during fly-back


SIM Scanner

156 msec/frame500 frames 80ms Tornado bleach

Bleach is completedwithin one frame and recovery starts.

Pyramidal Neuron Cell

FluoView FV1000 Transmitted Overview

FV1000 Components Transmitted light detection system suitable for visible

to near IR

FluoView FV1000 Laser Overview

• Basic SpecificationsFV1000 Components

Laser Combiners

Original Combiner

Current Dual Combiner Current Single Combiner


Spectral Un-mixing Applications

Spectral Un-mixing

CFP, GFP

GFP, YFP

Auto-fluorescence background removal

Separate dyes with overlapping excitation spectra

Variable Band Pass Filter Optimize detection band widthNew dyes, e.g.quantum dots,GFP variants

Quantitative Fluorescence Imaging FRET analysis free of bleed through


• Basic SpecificationsSpectral Detectors

Ch1 Ch2 Ch3

Ch4

Grating

Dichroic mirror

Barrier filter

Slit

ConfocalPinhole

Optional filterdetector

HV / Laser power was adjusted to get same intensity level

HV ： 600V 473nm ： 2% HV ： 600V 559nm ： 1.3%

HV ： 900V 473nm ： 2% HV ： 900V 559nm ： 1.3%

GaAsP

SpectralHigh HV

NEW VISIBLE (confocal) GaAsP Detectors

Comparison of Signal to Noise at Similar Level of Brightness

HV ： 600V 473nm ：13%

HV ： 900V 473nm ：13%

Visible GaAsP DetectorSpectral DetectorHigh HV(490 － 590nm)

Brain slice sample treated by SCALEVIEW-A2

Excited singlet state transition to long lived excited triplet stateROS and chemical modifications lead to destruction of fluorochrome

Reduce excitation energy. Increase sensitivity.

Photobleaching


Filter Detection

Integrated Signalis measured.

Band width dependent on selection of band pass filter.

Wavelength, nm

Rel

ativ

e F

l In

tens

ity


Spectral DetectionFluorescence SpectralProfile ( scan) is acquired.

Band width dependent on slit width setting.

Wavelength, nm

Rel

ativ

e F

l In

tens

ity


Spectral Detectors Lambda Scan

XYλ ScanExcitation 458nmDetection 480nm-660nm2nm Steps

Emission Spectrals

0

50

100

150

200

250

475

486

497

508

519

530

541

552

563

574

585

596

607

618

629

640

651

662

673

A B Detected signal (=A+B)


Spectral Detectors Lambda Scan


Spectral Un-mixing Superior Spectral Separation

2nm ResolutionAlexa 488 and YOYO-1 can be distinguished

10nm ResolutionAlexa 488 and YOYO-1 cannot be distinguished

Brainbow fish

FluoView FV1000 OverviewVisible Confocal Features

• Upgradeability and flexibilityFV1000 is totally modular. Additional lasers can be added to the combiner. A 4th fluorescent channel is available. Visible GaAsP available.A fiber port is available for attachment to third party built

accessories.SIM Scanner can be added. TIRF can be added.

Most upgrades can be done in the field with minimal downtime.

Fluorescent and Confocal Applications

FRETFRAPFLIPFLIMFCSTIMELAPSEINDICATOR DYES(CO)LOCALIZATIONBIOSENSORSTIRFPHOTOCONVERTING

GCAMP2

COMBINING RATIOMETRIC INDICATOR IMAGING WITH FRET IMAGING

INTERPLAY BETWEEN CALCIUM AND cAMP

FURA-2 AM AND EPAC1-CAMPS

Roe et al.

KAEDE - PHOTOCONVERTING FLUORESCENT PROTEIN

Photoactivatable GFP

Multiphoton Imaging

Mouse Cerebral Cortex Image provided by: Kei Eto, Hiroyuki Inada, Yusuke Takatsuru, Hiroaki Waki, Tomomi Nemoto, and Junichi Nabekura, National Institute for Physiological Sciences, National Institutes

of Natural Sciences, Japan

Visible light confocal Multiphoton excitation

Layer V neurons800m deep

Why multi-photon microscopy?

• MPE microscopy is an optical sectioning technique in the same way that confocal microscopy is.

• Optical sections can be combined into three dimensional images just as they are in confocal microscopy.

• MPE microscopy usually causes less photodamage than confocal microscopy.

• Reduced scattering losses from the illuminating beam because it is at a much longer wavelength.

• MPE microscopy produces better images in thick, highly scattering specimens than confocal microscopy.

MPE microscopy increases signal to noise leading to improved resolution/contrast.

MPE Imaging

MOUSE TRANSGENICEYE EXPRESSINGtdTOMATO

960nm

Z-series

Optical sectioning

DEEP

Comparison of Multiphoton Excitation with Confocal

MPE can image deeper than confocal e.g. into tissue where light scattering occurs

MPE can image UV probes without need for UV illumination

MPE allows localised interaction with sample and thus less photo-bleaching and photo-toxicity (excitation in focal volume only).

MPE can image live cells and tissue for longer than confocal as a result of the reduced photo-toxicity, e.g embryonic development

MPE has similar resolution due to same emission wavelength as confocal

Some endogenous pigments (melanin) absorb NIR (MPE)

Transgenic mouse eyes expressing tdTomato

MULTI-PHOTON MICROSCOPY: APPLICATIONS AND THEORY … and MP… · multi-photon microscopy:...

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