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Confocal Microscopy

Dr. Serge Arnaudeau Bioimaging Core Facility Geneva

Wide-field microscope
in focus

Light source

Sample (object plane)


Viewing plane (image plane)

• only one plane in focus • but all the planes contribute to the image

The pinhole
in focus pinhole Light source

Sample (object plane)


Viewing plane (image plane)

Photons passing through the pinhole are coming exclusively from the focal point of the objective

Depth of field depends on pinhole size

small pinhole in focus objective

large pinhole

small pinhole most of the photons coming from out of focus planes are rejected and do not contribute to the image

Confocal microscope principle x pinhole Light source detector Transmissive design Sample objective (object plane) y pinhole objective • “conjugate focal planes” • illumination and detection of the same focal point • need to displace the sample in x and y to construct an image .

What is fluorescence? Excited state Absorb high energy photons Emit lower energy photon Ground state In one-photon excitation λex < λem (Stoke’s shift) .

How does a fluorescence microscope work? Emission filter Light source Dichroic filter objective Excitation filter sample .

Epitaxial confocal microscope for fluorescence photomultiplier tube barrier filter pinhole • use of the same objective for illumination and detection • use of a laser source to avoid the use of a pinhole in illumination • use of a PMT to make photon counting for each focal point • use of galvanometric mirrors to XY scan the field of view objective dichroic mirror Laser Source • use of a stepping motor in the Z direction to make optical slices in the sample Focal point .

Advantage of fluorescence confocal microscopy • ability to control depth of field • elimination or reduction of background information away from the focal plane • capability to collect serial optical sections from thick specimens A section of mouse intestine imaged with both confocal and non-confocal microscopy .

488. 633 nm Scanning head System electronic rack . 477. 561. 514.How big is a Laser Scanning Confocal Microscope ? Laser module 405. 458.

Alexa546 …) • 561 nm DPSS laser (Texas red. GFP. CFP…) • Argon ion gas laser with 458 nm (CFP…) 488 nm (FITC. Cy3. Cy5 …) . Alexa568 …) • Helium neon 633 nm gas laser (TOTO3. Alexa488 …) 514 nm (YFP…) • Helium neon 543 nm gas laser (TRITC.LASER Light Amplification by Stimulated Emission of Radiation • High intensity • Spatial and temporal coherence • Monochromatic • Focused Lasers installed in our Laser Scanning Microscopes • 405 nm Diode laser (DAPI.

Wide-field illumination cone versus point scanning of specimens • Wide-field microscope : entire depth of the specimen over a wide area is illuminated • Confocal microscope : the sample is scanned with a finely focused spot of illumination centered in the focal plane .

Beam scanning Majority of laser scanning microscopes : single beam scanning Laser spot To scan the specimen in a raster pattern. The scanning speed is limited by these mirrors. Good image quality but not fast enough to resolve transient physiological signals Only confocal microscopes which use acousto-optic deflectors can scan at speeds of 30 frames/s . the Laser Scanning Microscope uses a pair of computer controlled galvanometric mirrors.

Photomultiplier Tubes (PMT) Anode Photocathode Window Incident light Dynodes Side on design Gain varies with the voltage across the dynodes and the total number of dynodes With typically 9 dynodes. gain of 4x106 can be achieved .

1 0 0.01 100 200 300 400 500 600 700 800 900 1000 0V 50 V offset Wavelength (nm) Low quantum efficiency and low dynamic range but very fast response time . quantum efficiency.Photomultiplier Tubes (PMT) The spectral response. sensitivity. and dark current of a photomultiplier tube are determined by the composition of the photocathode 100 Quantum Efficiency (%) 255 10 600 V 800 V Gray levels (8bits) gain 1 128 0.

Scanning speed influences image quality Muntjac cells – Alexa 555 anti OX Phos complex V inh prot 2 µm pixel dwell time 3.2 µs pixel dwell time 25.6 µs Better signal to noise ratio with low scanning speeds but samples are more exposed to the laser beam .

Scans averaging reduces noise Muntjac cells – Alexa 488 phalloidin 10 µm Average of 2 scans Average of 8 scans But greatly reduce the frame rate .

Airy disk and Resolution Due to diffraction. the image of a point source of light in the focal plane is not a point it’s actually an Airy diffraction pattern Airy diffraction pattern Airy disk The resolving power of an objective determines the size of the Airy diffraction pattern formed .

61 λexc /NA(obj) with NA(obj) = n sinα α n = medium refractive index α = objective angular aperture .Airy disk and Resolution The radius of the Airy disk is given by : r(Airy) = 0.

Airy disk and Resolution Rayleigh criterion for lateral resolution : the center of one airy disk falls on the first minimum of the other airy disk intensity contrast resolved Rayleigh criterion unresolved .

Pinhole and Resolution Confocal pinhole size = diameter of the Airy disk (1 Airy unit) 84% of in focus light pass to the detector Airy disk units are a convenient way to normalize confocal pinhole size : Pinhole size = 1 Airy unit = best signal to noise ratio .

Pinhole and Resolution Confocal fluorescence : pointwise illumination + pointwise detection narrower Point Spread Function / widefield microscopy Axial PSF intensity profiles Increase in lateral resolution rlateral = 0.4 λexc / NA widefield confocal confocal lateral resolution > widefield lateral resolution .

4 = 139 nm raxial = 1.Pinhole and Resolution Confocal PSF The PSF is elongated in the axial direction Axial resolution of an objective is worse than its lateral resolution Axial resolution : raxial = 1.4 x 488/1.4 λexc n / NA2 λexc = excitation wavelength n = medium refractive index NA = objective’s numerical aperture For an oil immersion objective with 1.515/(1.4 x 488 x 1.4)2 = 528 nm (in theory for very small pinhole size) .4 NA using the 488 nm laser line rlateral = 0.

8 µm) Pinhole : 0.5 µm) Better Z discrimination with small pinhole size but needs strong signals .5 AU (optical slice ~ 0.Resolution depends on pinhole size Muntjac cells – Alexa 488 phalloidin 10 µm Pinhole : 1 AU (optical slice ~ 0.

Optical sectionning X Y 5 4 5 4 3 2 1 3 z 2 Z y 1 x 3D reconstruction .

Sampling in confocal microscopy x y Pixel on the image The image is built as the laser moves on the sample Zooming is produced by slower movement of the laser on a reduced area : no pixelization effect even with very high zoom z x y Voxel on the sample .

Sampling in confocal microscopy 512x512 zoom 1 260 nm/pixel 512x512 zoom 2 130 nm/pixel 512x512 zoom 4 65 nm/pixel 20 µm 10 µm 512x512 zoom 8 512x512 zoom 16 16 nm/pixel 5 µm 33 nm/pixel 2 µm 1 µm Muntjac cells Alexa 488 phalloidin Alexa 555 anti OXPhos complex V inh prot TO PRO-3 .

3 x fhighest (to compensate low-pass filtering) .Y resolution of the optics Sampling is sufficient when there is enough pixels to separate two adjacent Airy disk Nyquist theorem : to reconstruct a sine wave : fsampling = 2 x fwave In imaging.Sampling in confocal microscopy What is the zooming factor limit? This is linked to the X. frequency = spatial frequency fsampling = 2.

3/rlateral undersampling > Pixel size ~ rlateral/2.3 > oversampling .Sampling in confocal microscopy The highest frequency to be sampled in the CLSM is imposed by the optical system : fhighest = 1/resolution To fulfill the Nyquist criterion : fsampling = 2.

Sampling in confocal microscopy Critical sampling distances @ 500 nm (for pinhole = 1 AU values by 50%) .

Ideal emission separation PMT 1 λτ Red emission filter λτ Dichroic beamsplitter PMT 2 Green emission filter λτ .

Crosstalk problems Most of the time there is some overlapping between fluorophores emission spectra Example of FITC and TRITC If the fluorescence signals are not taken sequentially : some of the green fluorescence is assigned to the red channel Using 488 nm and 543 nm lines : 22% overlap .

multitrack configurations allow sequential acquisition of lines (or frames) by very fast switching of the laser lines by means of AOTF Minimize crosstalk between channels More accurate quantification in co-localization experiments .Crosstalk problems To avoid bleed-through of one fluorescence in another channel.

Spectral separation When the emission spectra of the fluorophores are very close : Spectral detector (like the Meta detector) allow the record of the emission spectra of each pixel of the image Example of latex bead with narrow fluorescences in the core and the ring acquired with the spectral detector (Meta) Image serie of the bead at different wavelengths .

Spectral separation Selection of the different fluorescences (core and ring) Fluorescence separation after software unmixing .

FRAP Fluorescence Recovery After Photobleaching bleach recovery Use of the high power of the laser to photobleach a defined region of the sample The recovery of fluorescence in this region indicates any kind of movement (diffusion or transport) of fluorescent molecules The recovery time (half-recovery time) indicates the speed of this mobility .

FRAP experiments FRAP-recording for 40 min (1 frame/min) .

mitochondria (MitoTracker Red). some mitochondria are marked for photobleaching Very high control of the scanner by the DSP (Digital Signal Processor) to position the laser beam and choose ROI of any shape Bleaching of marked mitochondria with pinpoint accuracy (left) Merged images of mitochondria before and after photobleaching :bleached portions appeared in red (right) .Photobleaching Bovine endothelial cells actin filaments (BODIPY FL).

Other beam scanning techniques Multiple beam scanning : the Nipkow disk Disk rotation One way to increase the scanning speed is to increase the number of scanning spots. The spinning disk with pinholes was introduced into a microscope by Mojmir Petran in 1968. .

Improvement of the Nipkow disk principle in the YOKOGAWA scanhead Laser beam Collector disk Microlenses (20 000) CCD camera Aperture disk Dichroic mirror Pinholes (20 000) Objective specimen .

Nipkow disk confocal microscope facilitate studies of ligth-sensitive processes Cell cycle in Drosophila Embryo expressing GFPHistone Dr. Caetano Gonzalez EMBL .

Nipkow disk confocal microscope facilitate studies of fast processes Ca2+ waves in cardiomyocytes loaded with fluo-3 Dr. Marisa Jaconi Geneva Image capture at 33 Hz using an intensified camera (Coolsnap Cascade from Photometrics) .

Other beam scanning techniques Slit scanning : a new approach in confocal microscopy •The circular laser beam is transformed to a line which scan the sample in only one direction •The emitted fluorescence of that line passed through a confocal line pinhole •This line (512 pixels) is detected by a ultrafast line CCD detector Scan speeds of 100 frames/s can be achieved .

Advantage of the LSCM : the line scan mode Fluo-3 [Ca2+]i (nM) 250 10 μm 125 0 Fura-red [Ca2+]i (nM) 10 μm 150 75 0 200 ms Spatially restricted. but very fast (1 line/2ms) .