Optical Microscopy Flashcards
(19 cards)
Properties of Visible Light
Visible
Easy to produce and detect
Non-ionising (gentle)!»_space; between wavelengths of 400nm VIOLET to 700 nm RED
Illumination Methods (x2)
Critial illumination
- 1 condenser lens
- image of light source in sample
Kohler illumination
- 2 condenser lenses
- uniform illumination (not most photon efficient, loses some)
PLAN APO
60X/1.40 Oil
infinity/0.17
WD 0.21
Flat-field corrector - abberation corrector
Lateral magnification - numerical aperture NA
tube length - coverslip thickness
working distance
define Resolution
the minimum distance at which 2 points can be distinguished
Rayliegh criterion = resolving power
Higher NA means?
The microscope can collect more light, leading to better resolution
d = lamda/ 2n sin theta
Abbe diffraction limit
d = resolvable distance
lamda = wavelength
n = refractive index
theta = half angle of collection
NA = n*sin theta
Phase contrast
differential interference contrast (DIC)
Phase:
- refractive index difference causes delays in light rays (phase difference)
- thin unstained samples, difficult to see with brightflield (cells)
DIC:
- phase difference from local changes in refractive index
- only works with glass, no tissue culture plastic
Why fluoresence microscopy?
- rejection of relected light > imaging inside cells and tissues
- tagging of specific components of the cell > high specificity and contrast
- single molecule sensitivity
- visible wavelength > less damage
wide-feild vs confocal vs multiphoton
widefield > out-of focus blur, photobleaching (easy to set up, fast image collection)
confocal > pinhole discards all light coming from above and below the focal plane (requires more equip + expertise, slower)
multiphoton > doesn’t require pinhole, flour only excited at the focal spot, also eliminates blur, longer wavelengths (red light) penetrate deeper into the sample, BUT lower res
lightsheet microscopy
excitation from the side by a second objective, no out-of-focus or photobleaching, used with larger samples ZEBRAFISH
fluorescent dyes vs protein labels
dyes:
- small, bright, photostable
- often toxic
- thick tissues can be problematic!
proteins:
- in vivo live imaging
- no issue with sample penetration
- needs cloning, complex, bigger size, redirect protein
Aspects of a digital image
- sampling rate (pixel size)
- image bit depth (dynamic range and contrast)
- file format
- image presentation
sampling rate
pixel size musy be chosen to ACCOMODATE the achievable resolution determined by the OBJECTIVE
inverse to pixel size
Nyquist Criterion
how you choose the sampling rate
the sampling frequency required to represent the true identity of the sample
you MUST SAMPLE at least 2.3 TIMES the HEIGHEST SIGNAL FREQUENCY to avoid missing details
i.e. pixel size should be 2.3 times smaller than the expected resolution
1nm pixel to resolve 2.3nm features
Bit depth
= how many binary digits (bits) are used in each pixel to store the intensity value
1 bit gives 2 choices (on/off)
number of grey levels = 2^bit depth
2^8 is enough
bit depth determines the resolvable intensity changes in an image
Histogram
graphical representation of the individual pixel grey values
information about which portion of the dynamic range we are using
want to cover the whole range without clipping at exactly black and white
Super-resultion techniques
Projection of a ____
_____ control of ___ states
spatial illumination pattern
> structured illlumination microscopy (SIM)
> stimulated emission depletion microscopy (STED)
stochastic, off/on
> Localisataion microscopy!
» stimulated emission depletion
» single molecule localisation microscopy
FLIM quantifies FRET
Fluoresence lifetime imaging
> measure decay in each pixel of the image
> map fluorophore microenvironment (ion concn, pH, temperature etc.)
Forster resonance energy transfer
- energy transfer between two molecular, emission and excitation overlap
- quantity the lifetime of the donor for FLIM
= molecular ruler
