Basics of confocal imaging (part I)

Transcription

1 Basics of confocal imaging (part I) Swiss Institute of Technology (EPFL) Faculty of Life Sciences Head of BIOIMAGING AND OPTICS BIOP

2 Lateral resolution BioImaging &Optics Platform Light microscopy Transmission Fluorescence d min 1.22λ NA obj + NA cond = 0 = 0.61 λ 0 rairy NAobj Ernst Abbe Lord Rayleigh Depth of field (Axial resolution) d tot = n λ NA 0 2 obj 2

3 Imaging of thick specimen wide-field confocal out of focus light is bluring the image and thus reducing the contrast/resolution 3

4 Overview 1. History of confocal 2. Confocal principle 3. Main components 4. Important parameters 4

5 1. History of confocal Historical overview First confocal First spinning disc First laser confocal 5

6 Historical overview BC (before confocal) spinning disk for image dissection (Nipkow) AD (after [confocal] development) stage scanning confocal (Minsky) invention of the laser tandem-scanning confocal microscope (Egger, Petráň) 1980s - development of personal computer commercial laser scanning confocal 6

7 First confocal microscope Transmission light Marvin Minsky,

8 First spinning disc confocal Reflected light Egger, Petráň [Petrah nyu],

9 First laser confocal Reflected light Davidovits, Egger,

10 2. Confocal principle How it works Scanning mechanisms Modes of operation Confocal vs wide field 10

11 How it works Focal plane Out-of-focus plane Image plane Problem: Wide-field image contains significant amount of blur due to out-of-focus contribution 11

12 How it works Pinhole Out-of-focus plane Image plane Focal plane Problem: Wide-field image contains significant amount of blur due to out-of-focus contribution Solution: confocal or deconvolution 12

13 Imaging point by point Most of the light is rejected by the pinhole Intensive light source: laser Sensitive detector: PMT Scanner Computer 13

14 Wide-field BioImaging &Optics Platform Illumination Köhler illumination illumination source is in a fourier plane to the sample plane -f f confocal Critical illumination illumination source is in a conjugate plane to the sample plane -f f 14

15 X-Y scanning mechanisms To obtain 2D image the specimen is scanned line by line Unidirectional or bidirectional scan Beam scanning is mostly used 15

16 Z scanning mechanisms To obtain 3 D image of the specimen, it is necessary to move the excitation focus not only in XY direction but also in Z direction Move objective Z drive (large range, tens of nm accuracy). Zeiss, Olympus Piezo holder (small range but nm accuracy). PerkinElmer UV Move stage Piezo (high accuracy, but low stroke). Leica Galvo (large stroke, high accuracy). Leica 16

17 Modes of confocal Reflection Mostly used in materials sciences Useful for settings optimization Fluorescence Main mode for imaging in biology Also used in materials sciences 17

18 Additional possibilities Transmission For amplitude samples DIC For phase samples Laser polarized Koehler illumination important Additional detector required (PMT) No pinhole - images not confocal 18

19 3. Main components Lasers Photomultiplier Spectral detection AOTF Filters, dichroics Objectives 19

20 Lasers Light Amplification by the Stimulated Emission of Radiation Gas laser Solid state laser Diode laser Diode pumped solid state laser Properties of laser-light Linear polarization Coherence (temporal and spatial) Narrow spectra (several nm) High intensity (up to several W) 21

21 Typical laser lines Gas lasers: HeCd 442 Ar ArKr HeNe Diode or DPSS (More compact and easy to operate) 22

22 Photomultiplier Quantum efficiency up to 12% Gain up to several millions Nonlinear gain on voltage 23

23 Spectral detection Diffraction grating or prism decomposes fluorescence light into spectrum 24

24 Acousto Optical Tunable Filter (AOTF) Microsecond temporal resolution High transmission Intensity control possible Blanking outside scan area 25

25 Filters, dichroics AOTF instead of excitation filters Emission filters: LP, BP Dichroic (dichromatic): single, multiple Should match the fluorophore 26

26 Choice of objective Zeiss 63x/1.2 W Korr wd: 0.24 mm; thick specimen Zeiss 63x/1.4 Oil wd: 0.18 mm; thin specimen Match refractive index of sample and immersion media Use objectives with correction collar For confocal imaging objective magnification is not so important, only NA and degree of objective correction matters 27

27 Typical LSCM system 28

28 Scanning mechanism 29

29 Typical LSCM system 30

30 4. Important parameters Pixel dwell time Zoom and pixel number Laser line and power Pinhole size 31

31 Pixel dwell time How much time signal is collected at every pixel Very small values, normally in microseconds range Defined either directly or indirectly by scan frequency and image size Example: 512x512 pixels, 400 Hz - dwell time sec Important characteristic for signal intensity 32

32 Zoom and pixel number Pixel size is defined by zoom and pixel number To change pixel size 1. Change zoom - image size changed, number of pixels constant 2. Change number of pixel - image size constant, pixel size changed 512x x x

33 Laser line and power Laser should correspond to fluorophore excitation spectrum Excitation outside the absorption peak is still possible This might be useful to decrease auto fluorescence, but be careful with cross excitation Avoid detector saturation, use gain offset to adjust signal Avoid fluorophore saturation 34

34 How to choose pinhole size Pinhole is measured in Airy units 1 AU - diameter of the first minimum of the Airy disc Sectioning/signal optimum at 1 AU Pinhole diameter: d < 0.7 AU: optimal sectioning but very low signal 0.7 < d < 1 AU: some reduction in sectioning quality but significant increase in signal d > 1 AU: rapid decrease of optical sectioning quality 35

35 Summary Most of the light is rejected by the pinhole. Specimen with low florescence signal in widefield is very difficult to image in confocal. For confocal microscope objective NA and degree of correction is much more important than the magnification of the objective. Pinhole with the size of 1 AU is a good trade off between signal intensity and sectioning quality. 36

36 Confocal vs. widefield Confocal Wide field Detector efficiency 3-20% (PMT) 60-80% (CCD) Optical sectioning yes no Signal level low high Frame rate low high Power density very high low Optimal usage Main limitation one plane in thick specimen Fluorophore saturation Scattering sample defocus signal thin sparsely stained specimen Out-of-focus light 37