NSC837 Lecture 3 - Sequential imaging, laser light bleed through, confocal scanners, PMT detectors

fluorescence crossover

fluorescence from shorter wavelength flurochrome crosses into longer wavelength detection channel (prevent with sequential imaging)

sequential acquistion

illuminate with different lasers separatley to avoid crossover associated with simultaneous acquisition

laser light bleed through

wide band pass filters (ch1 BP 475-525nm would allow 488nm laser light through) (ch2 BP 505-560nm would allow 543nm light through)

confocal laser scanning microscopy

optically reduces the amount of out-of-focus light fromt he image

pinhole apeture blocks out of focus light by

controlling size of pinhole apeture can impact the image

where does term galvo (or scan) mirrors come from

the name of the motor that moves the mirrors

photo multiplier tube (PMT)

used as photodetectors (image formation)

confocal scanning

X and Y Scan Mirrors (galvo mirrors) - controls the exact position of the laser within the field of view of the sample

AOTF - microsecond shuttering and intensity control of the laser (turn of / on intensity of laser)

2 types of point scanning

1) uni-directional -standard scan is left to right, more accurate

2) bi-directional - rapid scanning, but can produce jagged edges within the image (scanning mirrors have to slow down/speed up before/after turns). This has been fixed by having galvo mirrors start outside of field of view

bi-directional better for live cells (moving)

resonance scanning

Probably won’t need for fixed but maybe for live

Some scopes only have galvo some have both where you can choose

multi-point scanning confocal (spinning disk)

two spinning disks with dichroic mirror between the two disks

camera detection instead of PMT

can illuminate more than one point at a time

what problem arises with multipoint-scanning confocal

missing laser- only a little bit of laser going in holes

original not good for biology but is good for lasers

advantages of spinning disk confocal

1) Speed - depends on spinning disk rate and camera readout rate (less res. for spinning)

2) sensitivity - quantum efficienty of cameras about 95% (PMT is 25-50%)

3) reduced photobleaching - low intensity longer exposure (inverse for point scanner)

point scan confocal advantages

1) optical sectioning- adjustable pinhole apeture; optimize for each objective and wavelength . Spinning disk does not have adjustable pinhole apeture, optimized for 100x objective, and green excitation only. Out of focus light can cross into other pinholes

2)multi-color imaging - multiple PMT detectors, simultaneous detection. Spinning only has single ccd camera and sequential detection

3) region of interest scanning/bleaching - scan mirrors/AOTF permit regional scan/bleach. Spinning disc is limited to FOV, no point bleaching

When might spinning disck be useful?

live cell imaging over time and rapid 3d imaging over time

reason: reduced photobleaching and faster scan speeds

photomultiplier tubes design

includes a photocathode, a focusing electrode, an electron multiplier (dynode), and an electron collector (anode)

photocathodes types

multi-alkali photocathode detector (standard) - highly sensitive in UV range and not as easily damaged

GaAsP (gallium arsenide phosphide detector) - highly sensitive throughout visible wavelength range. Can be easily damaged by intense light

PMT dynamic range

the range of detectable intensities that the PMT can record

dimmiest - below is zero. This is the offset value

upper limit (brightest) - above this is saturation. Determined by gain

what do PMT detectors convert fluorescence density into

a digital intensity value

bit depth

bit depth determines the number of increments or intensity levels between zero and saturation for a PMT

actual intensity value is recorded by the PMT is determined by the bit depth

doesn’t matter for pretty pics but does matter for quantifying

Look-Up Table (LUT) (color assignment)

image displayed on the monitor is not a “true” color image

color is a pseudo color chosen by the user. defined by LUT

It maps color intensity to th efluorescence intensity detected by the PMT

kalman averaging

high detector gain settings will create random bright spots or noise in the image

this is due to random electrons generated and detected within the PMT detecto (not true signal from sample)