Imaging Glossary
Microscopy Technology & Optical Systems
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4-1BB/4-1BBL
4-1BB (CD137) / 4-1BB Ligand (CD137L)
Activating immune checkpoint receptor-ligand pair that promotes anti-tumor immunity when engaged.
Acquisition Overhead
The time spent during image acquisition that is not collecting photons from the sample. This includes filter wheel changes, dichroic mirror switching, stage movements, and camera readout time.
System Performance
Airy Disk
The diffraction pattern formed when light from a point source passes through a circular aperture. Even a perfect lens cannot focus light to an infinitely small point—instead, it creates this characteristic pattern of concentric rings surrounding a central bright spot.
Resolution & Sampling
Camera Count
The number of cameras in a multi-camera detection system. More cameras enable more simultaneous channels, reducing filter wheel changes and acquisition time, but increase cost and system complexity.
Optical Path Components
Camera-Objective Matching
The process of selecting camera pixel size and objective magnification to achieve optimal Nyquist sampling while balancing sensitivity, field of view, and throughput requirements.
System Performance
Channel Count
The number of distinct fluorescence channels in the imaging experiment. More channels provide more information but increase complexity, potential for bleedthrough, and acquisition time (especially without multi-camera detection).
System Performance
Confocal Microscopy
An imaging technique that uses pinholes to reject out-of-focus light, enabling sharp optical sections through thick samples. Unlike widefield microscopy where out-of-focus regions contribute blur, confocal produces crisp images from specific focal planes.
Fluorescence Methods
Dichroic Mirror
An optical filter that reflects certain wavelengths while transmitting others, based on thin-film interference coatings. In fluorescence microscopy, dichroics separate excitation light from emission light, enabling detection of fluorescence while blocking the bright excitation source.
Optical Path Components
Dynamic Range
The ratio between the largest and smallest signals a camera can accurately capture in a single image. High dynamic range allows imaging of samples with both bright and dim features without saturating bright regions or losing dim ones to noise.
Cameras & Sensors
Emission Filter
A bandpass filter positioned between the sample and detector that transmits only specific wavelengths of fluorescence emission while blocking excitation light and unwanted fluorescence from other channels.
Optical Path Components
Equivalent Sensitivity
The principle that different camera/objective combinations can achieve similar practical sensitivity through compensating factors. A system with lower QE or smaller NA can match a 'better' system if other parameters compensate.
System Performance
Exposure Time
The duration for which the camera sensor collects photons for each image. Longer exposures collect more signal but slow acquisition; shorter exposures enable faster imaging but require brighter samples.
System Performance
Field of View
The basic unit of image acquisition in StrataQuest. A Field of View (FOV) is a single image tile captured by the scanner at a fixed position. Whole-slide images are composed of hundreds to thousands of FOVs arranged in a mosaic grid and stitched together. StrataQuest processes analysis FOV-by-FOV, with options for local (per-FOV) or global (cross-FOV) computation.
Filter Wheel
A motorized device holding multiple emission or excitation filters that can be rotated to position different filters in the optical path. While enabling flexible multi-channel imaging with fewer cameras, filter changes add time overhead to acquisition.
Optical Path Components
Fluorescence
The emission of light by a molecule that has absorbed light at a shorter wavelength. The difference between absorption and emission wavelengths (Stokes shift) enables spectral separation of excitation from emission for sensitive detection.
Fluorescence Methods
Fluorophore
Fluorophore / Fluorescent Probe
A molecule capable of fluorescence, characterized by its excitation/emission spectra, brightness (extinction coefficient × quantum yield), and photostability. Fluorophore selection determines signal strength, spectral separation, and imaging duration limits.
Fluorescence Methods
Frame Rate
The number of complete images a camera can capture per second, determined by sensor readout speed and interface bandwidth. Higher frame rates enable faster Z-stack acquisition but may require brighter signals to maintain adequate signal-to-noise.
Cameras & Sensors
Full Well Capacity
The maximum number of electrons a pixel can hold before saturating. Larger pixels typically have higher full well capacity, providing greater dynamic range and tolerance for bright samples before saturation.
Cameras & Sensors
Immersion Media
The material filling the gap between the objective front element and the sample or coverslip. The immersion medium's refractive index affects numerical aperture, aberration, and compatibility with different sample types.
Objective Properties
Magnification
The ratio of image size to object size. In microscopy, total magnification is the product of objective and eyepiece (or tube lens/camera) magnification. Higher magnification spreads the same light over more area, reducing signal per pixel while enabling finer sampling.
Objective Properties
Multi-Camera Splitter
An optical device that divides the image into multiple spectral channels using dichroic mirrors, directing each channel to a separate camera for simultaneous acquisition. Eliminates filter wheel changes and enables true simultaneous multi-color imaging.
Optical Path Components
Numerical Aperture
A dimensionless measure of the objective's ability to gather light and resolve detail, defined as NA = n × sin(θ), where n is the refractive index of the imaging medium and θ is the half-angle of the maximum cone of light that can enter the lens.
Objective Properties
Nyquist Ratio
The ratio of optical resolution to sampling interval, indicating whether the pixel size properly captures the detail the objective can resolve. A ratio of 2.0-2.5 is optimal; below 2.0 means undersampling (losing resolution), above 3.0 means oversampling (wasting pixels).
Resolution & Sampling
Nyquist Sampling
The principle that to accurately capture the optical resolution, you need at least 2 samples (pixels) per resolution element. Undersampling loses information that the optics provide; oversampling wastes pixels without gaining detail but may enable better deconvolution.
Resolution & Sampling
Objective Transmittance
The percentage of incident light that passes through the objective at different wavelengths. Critical for near-infrared (NIR) fluorophores where many objectives show significantly reduced transmission above 700nm.
Objective Properties
Pentaband Dichroic
A dichroic mirror with five (or more) reflection/transmission bands, allowing simultaneous excitation with multiple laser lines without mechanical switching. Enables true multi-color imaging with no dichroic change overhead.
Optical Path Components
Photons per Pixel
The number of photons collected by each pixel during an exposure, before conversion to electrons. This is the starting point for all sensitivity calculations and depends on sample brightness, collection efficiency, and pixel area.
System Performance
Pixel Matching
The process of selecting camera pixel size and objective magnification to achieve optimal Nyquist sampling. When pixels are too large, optical resolution is undersampled; when too small, sensitivity is sacrificed without gaining resolution.
Resolution & Sampling
Pixel Size
The physical dimensions of each light-sensitive element on the camera sensor, typically measured in micrometers. Smaller pixels enable finer sampling but collect fewer photons per pixel; larger pixels are more sensitive but sample more coarsely.
Cameras & Sensors
Point Spread Function
The three-dimensional diffraction pattern describing how a point source of light appears after passing through the optical system. The PSF characterizes the system's resolution and is used for deconvolution to computationally improve image quality.
Resolution & Sampling
Quantum Efficiency
The percentage of incident photons that are converted to measurable photoelectrons. Higher QE means more of the precious photons from your sample contribute to signal rather than being lost at the sensor.
Cameras & Sensors
Read Noise
Electronic noise added during the process of reading out the sensor, measured in electrons RMS. Read noise sets the detection floor—signals below the read noise level cannot be reliably distinguished from noise.
Cameras & Sensors
Resolution Limit
The minimum distance between two points that can be distinguished as separate objects, fundamentally limited by diffraction of light. No amount of magnification or better cameras can improve resolution beyond this limit—only higher NA or shorter wavelengths help.
Resolution & Sampling
Sample Area per Pixel
The actual area of the sample captured by each camera pixel, determined by pixel size divided by magnification. This metric is critical for understanding throughput, sensitivity trade-offs, and proper Nyquist sampling.
System Performance
sCMOS Camera
Scientific Complementary Metal-Oxide-Semiconductor cameras combining low read noise, high speed, large field of view, and high quantum efficiency. sCMOS has largely replaced CCDs and EMCCDs for fluorescence microscopy.
Cameras & Sensors
Sensitivity Trade-offs
The interplay between optical and detector parameters that determines how efficiently the system converts sample fluorescence into detected signal. Understanding these trade-offs enables optimal system design for specific applications.
System Performance
Sensor Area
The total active imaging area of the camera sensor, determining the field of view at a given magnification. Larger sensors capture more sample area per frame, directly affecting imaging throughput for large specimens.
Cameras & Sensors
Sequential Acquisition
Imaging each fluorescence channel in sequence rather than simultaneously, eliminating spectral bleedthrough at the cost of additional time for separate exposures. Essential when emission spectra overlap significantly.
Fluorescence Methods
Signal-to-Noise Ratio
The ratio of detected signal to the uncertainty (noise) in that measurement. Higher SNR means clearer images and more reliable quantification. SNR determines whether dim features like single mRNA spots can be detected.
System Performance
Simultaneous Acquisition
Capturing multiple fluorescence channels in a single exposure using spectral separation and multiple detectors. Eliminates filter wheel switching and enables true co-temporal imaging of dynamic samples.
Fluorescence Methods
smFISH
Single Molecule Fluorescence In Situ Hybridization—a technique for detecting and counting individual mRNA molecules in fixed tissue using multiple short fluorescent probes per transcript. Each mRNA appears as a diffraction-limited spot that can be identified and counted.
Fluorescence Methods
Spectral Bleedthrough
Spectral Bleedthrough / Crosstalk
Unwanted detection of fluorescence from one channel in another, caused by overlapping emission spectra, imperfect filter separation, or cross-excitation of fluorophores by off-target wavelengths. Bleedthrough creates false colocalization and complicates quantitative analysis.
Fluorescence Methods
Throughput
The rate at which sample area is imaged, typically measured in mm²/sec. Throughput integrates exposure time, frame rate, field of view, and acquisition overhead into a single metric for comparing system performance.
System Performance
Tissue Clearing
Chemical treatment to make tissue optically transparent by removing light-scattering components and matching refractive indices throughout the sample. Enables imaging through thick tissue volumes without mechanical sectioning.
Fluorescence Methods
Total Acquisition Time
The complete time required to acquire all images for a sample, including exposures, readout, mechanical movements, and any overhead. This is the bottom-line metric users care about for workflow planning.
System Performance
Tube Lens
The lens that focuses parallel light from an infinity-corrected objective to form an image at the camera sensor. The tube lens focal length determines the effective magnification of the system.
Optical Path Components
Water Dipping Objective
An objective designed to be dipped directly into aqueous solution without a coverslip, providing the combination of high numerical aperture and long working distance that is impossible with conventional immersion objectives.
Objective Properties
Working Distance
The distance between the front element of the objective and the sample surface when in focus. Working distance is critical for thick samples, specialized mounting, and avoiding contact between the objective and sample.
Objective Properties
Z-Planes
The number of focal planes captured in a Z-stack, determining the axial extent and resolution of 3D imaging. More planes provide finer Z-sampling and deeper coverage but increase acquisition time and data volume.
Fluorescence Methods
Z-Stack
A series of images captured at different focal planes through the sample, enabling 3D reconstruction, maximum intensity projections, or optical sectioning of thick specimens. Z-spacing should match optical section thickness for optimal sampling.
Fluorescence Methods