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Basic Quantitative MicroscopyPractical Pitfalls in Image Acquisition

Johanna Dela Cruz2 March 2016

What are my imaging goals?

• resolution• contrast• remove the greatest amount of out-of-focus light• maintain a detectable signal• minimize cell phototoxicity• minimize fluorophore bleaching• speed• multidimensional• image area

How should I image my sample?

• Brightfield illumination (BF)• Phase contrast (PC)• Differential interference contrast (DIC)• Darkfield (DF)• Polarized light

Transmitted light microscopy

• Standard Widefield• Confocal/multiphoton/TIRF/PALM/STORM/

STED

Incident or reflected light/epi-

illumination


Transmitted light microscopy:

Stained/Unstained samples

Halogen lamp

condenser

Specimen

objective

ocular


Incident or reflected light/epi-

illumination: Metals; opague;

Fluorescence-labeled

Halogen lamp

condenser

Specimen

objective

ocularMercury lamp

Dichroic Filter


Transmitted light microscopy:

Stained/Unstained samples

• Standard Widefield fluorescence• Confocal/multiphoton/TIRF/super-

resolution

Incident or reflected light/epi-illumination:

Metals; opague; Fluorescence-labeled

Brightfield Brightfield Darkfield

BF: contrast generated from changes in light absorption, refractive index or color

DF: scattered light caused by optical discontinuities

Phase Contrast DIC FluorescencePC: contrast from

interference of light path lengths through sample

DIC: use of light-shearing prisms and polarized light to exaggerate minute differences in specimen

thickness gradients and refractive index

• Thin samples

• Optical sectioning of thick samples

Conventional Widefield or Confocal?

• Thin samples

• Optical sectioning of thick samples

Conventional Widefield or Confocal?

Widefield fluorescence microscopy

Confocal microscopy(optical slice projection)

fluorophore is excited with one wavelength of light and light of a different wavelength is

emitted

Fluorescence

Upright vs. Inverted Microscopes

- No fundamental difference in the ability to produce and channel light along various paths

- Image quality dependent on sample preparation, objective lenses, light source and wavelength, fluorophore filter set, detector

Sample Preparation“Garbage in = Garbage out”

• Non-fluorescent contrasting agents/stains – immunohistochemical• Fluorophores: extinction coefficient, quantum yield, photostability• fixed-cell imaging: fixation and permeabilization, signal enhancers• Immunofluorescence imaging: antibodies• Live-cell imaging: imaging media, background suppressors• Type of mountant used:

PVA vs glycerol-based Anti-fade agents and compatibility with fluorophores Coverslip sealant (nail polish, VaLaP)

Optical properties of the microscope that you need to know about

Objective lens – most critical component of a microscope• Magnification• Numerical aperture (NA): light-gathering ability

- resolving power- signal intensity/brightness ∞ (NA)4 / Mag2

Which would you choose?40x, 0.75 NA vs 40x, 1.3 NA60x,1.4 NA vs 100x, 1.4 NA

Manufacturer

Class/Special designation

Magnification

Numerical aperture, NA

Immersion medium

Specialized optical properties/ contrast method

Infinity-correctedCoverslip thickness

Color of objective text: contrast methodstandardpolarization/DICphase

Color coding of Immersion fluidoiloil/water/glyceringlycerin (glyc)water (W)

Color coding of Magnification4x/5x10x20x40x63x100x/150x

Correction collar• Cover glass thickness

correction• Different immersion• Different temperature

Coverglass thickness• 0.17: standard (#1.5)• 0 : w/o coverglass• – : insensitive

Resolution

• most important factor that determines image quality

• amount of detail you can see in an image

• Without a sufficiently high resolution, magnification is not possible without loss of quality.

Finest observable details ~ λ/2NA

Factors Affecting Resolution

• Objective-related: correction for aberration, NA• Specimen-related: coverglass, mounting medium, immersion

medium• Lighting: excitation wavelength, color range• System stability

Numerical Aperture

Olympus Primerhttp://olympus.magnet.fsu.edu/primer/java/nuaperture/index.html

NA = n sin(θ)n = refractive index

(1.0 -> air)Θ = angular aperture

0.25 NA

0.5 NA

0.95 NA

http://olympus.magnet.fsu.edu/primer/java/nuaperture/index.html

Numerical Aperture

40x/0.6 40x/1.3

Other features of the objective may prove more critical for a particular sample or application:

Why would anybody choose an objective of lower NA?

• Working distance: how far objective can focus into sample

• Design for use with/without coverslips

• Corrections for flatness of field, chromatic and spherical aberrations

• Transmission of specific wavelengths (UV, IR)

• Refractive index of immersion medium proportional to NAoil water air (decreasing NA)

• Brightness: which would you choose? 40x/1.2 W vs 63x/1.4 oil

major cause of the loss in signal intensity and resolution with increasing focus depth through thick specimens

Spherical Aberration

Increased by:• use of the wrong coverslip thickness• type of immersion oil • thick layer of mounting medium• presence of air bubbles in the immersion or

mounting medium• temperature change use of oil immersion on aqueous sample

L: at 0 um; R: at 35 um into sample

wavelength-dependent artifacts that occur because the refractive

index of every optical glass formulation varies with

wavelength

Chromatic Aberration

• Coverglass: protect specimen integrity and provide a clear window for observation

• Introduces chromatic and spherical aberration (loss of contrast) that must be corrected by objective lens

• Thickness: 0.13 to 0.22 mm; standard: 0.17 mm• Negligible for dry objectives with NA < 0.4; significant at NA > 0.65

• Effect of mounting medium

Coverglass and Mounting Medium

Matching Immersion Medium to Objective

• Immersion media: used to minimize the refractive index (RI) differences present in space between objective and sample includes substrate (glass coverslip) sample is on and imaging

medium (buffer) sample is in.

• NA partly depends on the RI of the immersion medium [NA = n sin(θ)]

• Consequences of Mismatched RIs: spherical and chromatic aberrations, loss of resolution, reduced scan depth in z, wasted time and effort

• important factor in microscope resolution

• Resolution limited to ~1/2 wavelength of illumination

• Shorter wavelengths (near UV) yield higher resolution

Wavelength

Know your fluorophores, excitation sources and filter sets

Excitation spectrum

Emission spectrum (fluorescence)


• Emission wavelength is independent of the excitation wavelength• emission intensity is proportional to the amplitude of the excitation wavelength,

Mercury arc light output


• Emission wavelength is independent of the excitation wavelength• emission intensity is proportional to the amplitude of the excitation wavelength,

Laser output at 405 nm

Multiple labeling

Choosing fluorochromes with well-separated excitation and emission spectra is critical

Emission Filters

Band-pass filters collect emissions in a specific range. The narrower the range of the band-pass filter, the better it can separate fluorochromes with close emission spectra.

Simultaneous vs Sequential Imaging

Simultaneous Acquisition: bleedthrough/cross-talk

Sequential Acquisition: well-separated

• Start at the lowest laser power possible, with a higher PMT voltage (>600 V) and gradually increase the laser power as required

• Averaging: better signal-to-noise ratio

• More light in plane of focus does not increase signal intensity: more out-of-focus fluorophores excited can lead to poorer z-axis resolution and increased photobleaching/phototoxicity

Fluorophore Signal

Sampling Frequency: Undersampling and Oversampling

Undersampling Good sampling Oversampling

Large pixels – low resolutionBrighter, less exposure,

less photobleaching

Too small pixels – excess of information,

dimmer, increased bleaching, waste of time & storage

Nyquist sampling

• CCD camera: averaging or binning increases S/N but decreases resolution• Binning: useful for large data sets or live-cell imaging, where binning and

shorter exposure times can be used to reduce phototoxicity• Binned images can be blurry: undersampling• using high NA objectives, making pixels smaller: does not always add

resolution because objects that are smaller than the wavelength of light cannot be resolved

• high zoom: smaller pixels, more data points, larger image files --specimen is oversampled, additional structural information not attained. For visible light and high NA objectives (>0.8) a pixel size of ∼0.1-0.2 μm is ideal.

Offsets and Detector

Saturation:

Avoid data clipping

Offset too high

Gain too high

Software Settings and Image Display

Original image Brightness/contrast enhanced

Gamma adjusted

• use the full dynamic range of the detector• Image display settings• best to display images in grey scale whenever possible• Dim features can be enhanced by modifying the display to a non-linear

LUT using the gamma factor • Display settings do not change the underlying data, however, these

manipulations should be mentioned in figure captions

Software Settings and Image Display

• The most critical components of the fluorescence microscope for quantitative imaging: objective lens, emission filter, and detector

• Wide-field microscopy: often inappropriate for quantitation because you collect emitted light from the whole sample depth without knowing the thickness of each cell or structure

• Confocal microscopy: more quantification-friendly for samples > 15–20 um in depth due to defined optical section thickness

• Deeper focal planes will show reduced signal intensity due to absorption and scatter, necessitating further, more complex corrections

Quantification of images—why is it useful and when is it appropriate?

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