Fluorescence Imaging Instruments: Microscopes, Plate Readers, and Spatial Platforms

Written by Emma Mason

Instruments for fluorescence imaging allow researchers to study a vast array of biological processes, typically aided by labels in the form of protein tags or synthetic dyes. Fluorescence imaging instruments range from gel documentation systems and multimode plate readers to high-plex spatial proteomics platforms and spectral flow cytometers. These technologies enable multiplex detection, quantitative analysis, and real-time visualization across diverse biological applications. Whether you want to multiplex western blot detection, run high-throughput cell-based assays, or perform intricate spatial profiling, innovative technologies are available to meet your every need. We spoke with Promega, Bio-Rad, Corporation, Bruker Spatial Biology, and BD to learn about some of the fluorescence imaging platforms on offer.

Key Milestones in Fluorescence Imaging

When Sir John F. W. Herschel first reported in 1845 that a naturally colorless solution of quinine exhibited an ‘extremely vivid and beautiful celestial blue’ when illuminated and observed under particular incidences of light, he probably never dreamt that his discovery would one day be harnessed to image biological samples¹.

However, Herschel’s finding caught the interest of other eminent scientists of the time, including Sir George G. Stokes, who established that certain atoms and molecules can absorb light at a particular wavelength and emit it at a longer wavelength after a brief interval². Stokes coined the term ‘fluorescence’ and gave his name to the Stokes shift, which describes the difference between a fluorophore’s excitation and emission maxima.

Fluorescence was first encountered in optical microscopy at the start of the 20th century, when August Köhler and Carl Reichert independently noticed that their samples generated unwanted signal when exposed to ultraviolet (UV) illumination. This observation was leveraged by the companies Carl Zeiss and Carl Reichert Optische Werke AG to develop the first fluorescence microscopes, which used UV illumination to visualize autofluorescence.

Over the years, fluorescence microscopy evolved, driven by the development of novel labeling methods. One of the earliest examples is Albert Coons’ use of a fluorescein isothiocyanate (FITC) labeled antibody to detect pneumococcal antigen in tissue sections³. Another is Osamu Shimomura’s discovery of green fluorescent protein (GFP), which was subsequently cloned and engineered to allow for protein labeling through genetic encoding^4-6.

Newer probes include various photoactivatable, photoconvertible, and photoswitchable fluorescent proteins, as well as fluorescent timers—proteins that change emission wavelengths over time, as first described by Terskikh et al. in 2000⁷. Additionally, a growing number of synthetic dyes is available, many of which address known limitations of existing fluorochromes.

• You can find detailed information on the optical properties and spectral profiles of more than a thousand fluorochromes in our Fluorescent Dye Database.

Importantly, fluorescence imaging is no longer limited to traditional microscopy-based applications. In fact, virtually all mainstream laboratory techniques have been adapted to employ a fluorescent readout for the improved sensitivity, capacity for real-time imaging, and significantly reduced hands-on time through multiplexing that it provides. The following are some leading examples of fluorescence imaging instruments.

Imaging Systems

GloMax® Galaxy Bioluminescence Imager System
A bioluminescence microscope with fluorescence and brightfield imaging capabilities

Promega’s GloMax® Galaxy Bioluminescence Imager System is a fully equipped microscope designed to visualize NanoLuc® Luciferase chemistries, which additionally supports fluorescence and brightfield imaging. “Using NanoLuc® luciferase technologies and the GloMax Galaxy, researchers can visualize tagged proteins at their endogenous levels in live cells,” explains Maggie Bach, Sr, Manager, Product Marketing. “Combining these readouts with fluorescence imaging increases spatial resolution, providing valuable context for protein localization.”

Validated applications include:

• Protein: protein interactions
• Protein localization and translocation
• Protein degradation and stability
• Ligand: protein interactions (target engagement)
• Targeted cell killing

ChemiDoc Imaging Systems
Gel and blot image documentation and analysis

Bio-Rad’s ChemiDoc Imaging Systems and accompanying Image Lab Touch Software offer best-in-class performance for gel and blot image documentation and analysis. “Our ChemiDoc Imaging Systems are highly sensitive, versatile, and intuitive instruments that enable fluorescence, chemiluminescence, and colorimetric detection,” reports Sahel Mohebbi, Field Application Specialist. “The ChemiDoc family also supports Stain-Free imaging for immediate visualization and verification of total protein on gel and membrane, allowing total protein normalization.”

The ChemiDoc family includes two specialized instruments with different fluorescence detection capabilities. “The ChemiDoc MP is our premium instrument, offering UV, RGB, far red, and near infrared channels, and enabling full-spectrum fluorescence multiplexing,” explains Kenneth Oh, Global Marketing Manager. “We also offer the ChemiDoc Go, a compact instrument featuring an advanced CMOS sensor for higher sensitivity, which is compatible with Bio-Rad’s unique and sensitive StarBright™ Blue 520 and 700 dyes for up to 2-plex fluorescence detection.”

GloMax® Discover Microplate Reader
A multimode microplate reader with advanced detection capabilities

While early microplate readers were generally limited to measuring absorbance, modern instruments have far greater functionality. In addition to UV-visible absorbance, Promega’s GloMax Discover Microplate Reader also permits fluorescence, luminescence, FRET, and BRET detection, using 6- to 384-well plates. “The GloMax Discover is a ready-to-use multimode plate reader developed with Promega reagent chemistries,” reports Bach. “It offers high sensitivity and a wide dynamic range to extend the linear range of your assay and can be either used as a standalone instrument or integrated into high-throughput automated workflows.”

Factors to consider when selecting a microplate reader:

• Flexibility across applications: Can the plate reader handle multiple detection modes, assay formats, and sample types?
• Ease of use and workflow integration: Does the plate reader have intuitive software, minimal training requirements, and compatibility with common assay kits to accelerate setup to results?
• Throughput and efficiency: Is the instrument compatible with different plate formats and can it be integrated with automation to streamline frequent or large-scale experiments?
• Sustainability and long-term value: Does the plate reader support modular upgrades, offer a broad assay menu, and come with reliable service to ensure more durable value over time?

CellScape™ Precise Spatial Proteomics
An automated high-plex cyclic immunofluorescence platform

The CellScape Precise Spatial Proteomics (PSP) platform from Bruker Spatial Biology automates high-plex cyclic immunofluorescence to generate quantitative spatial phenotyping data across whole slides.  “With EpicIF™ signal removal and a simple, flexible workflow, CellScape PSP makes it easy to run 60+ plex assays, whether with our validated VistaPlex™ kits or custom panels, while consistently producing publication-quality results,” says Tim Sindelar, Product Marketing Manager. “Its best-in-class resolution and dynamic range provide the reliable insights researchers need for biomarker validation and discovery.”

Key features of the CellScape PSP platform include:

• Simple assay design: Build custom panels at 60+ plex (4 markers/cycle).
• Modular workflow: Iteratively revisit stored samples for data-driven high-plex protein profiling while preserving precious tissue.
• Unmatched flexibility: Easily combine multiple fluorescence assays on the same sample, select the imaging mode that fits your study, and customize panels to your needs. Data outputs in open-standard OME-TIFF, making it simple to work with open-source tools, custom pipelines, or commercial analysis platforms.
• Quantitative spatial phenotyping data: High resolution and dynamic range for accurate cell segmentation and expression detection.

BD FACSDiscover™ Platforms Equipped with BD CellView™ Technology
Spectral flow cytometry and cell sorting with real-time image generation

In recent years, spectral flow cytometry has evolved to generate spatially informative images from cells in suspension, enabling real-time analysis and sorting using previously inaccessible dimensions of single-cell biology. “BD CellView™ Image Technology, integrated into the BD FACSDiscover™ A8 Cell Analyzer and the BD FACSDiscover™ S8 Cell Sorter, provides over 70 image-derived parameters per cell, including eccentricity, radial moment, total intensity, and delta center of mass,” says Rodrigo Pestana Lopes, Ph.D., Sr. Global Scientific Marketing Manager at BD. “As a result, researchers can now study processes such as protein trafficking, receptor localization, organelle activity, and cell-cell interactions using a spectral flow cytometer.”

BD CellView is a camera-free imaging system that brings real-time image generation to spectral flow cytometry by splitting a 488 nm laser beam into 104 frequency-tagged sub-beams. “As cells pass through the sub-beams, signals are collected and mathematically decoded using Orthogonal Frequency Domain Multiplexing (OFDM) to reconstruct high-resolution 2D images of each cell without the use of traditional optics,” explains Pestana Lopes. Detection includes forward (FSC) and side (SSC) scatter, axial light loss (ALL, producing brightfield-like images), and three fluorescence channels (534/46, 600/60, and 788/225). “Operating at speeds up to 12,500 events per second, BD CellView produces images comparable to 10-20x magnification in widefield microscopy and supports both label-free and fluorescent applications.”

Supporting Your Research – Design and Optimize Your Fluorescence Imaging Experiments

Planning a fluorescence imaging experiment? FluoroFinder’s tools help researchers design optimized antibody panels, select compatible fluorophores, and minimize spectral overlap across microscopy platforms. Use our Cell Types Tool to identify target antigens for your cell type of interest, then explore our Antibody Search function to find reagents validated for immunofluorescence applications. Our Fluorescent Dye Directory and Microscopy Spectra Viewer help you select dyes that match your instrument configuration and detection channels. To further support Microscopy experiments, FluoroFinder is developing:

• The Cyclic Immunofluorescent Panel Builder – a tool designed to streamline reagent selection, optimize staining rounds, and track the iterative process of designing Cyclic IF panels.
• Microscopy Spectra Viewer – configure your microscope setup and visualize fluorophore compatibility to design multiplex panels with confidence.

If you or a colleague is interested in contributing towards the development of these tools with ideas, beta testing, or feedback, please contact support@fluorofinder.com.

References:

1. Herschel JFW. On a case of superficial colour presented by a homogeneous liquid internally colourless. Philos Trans R Soc London 1845; 135: 143–145.
2. Stokes GG. On the change of refractibility of light. Philos Trans R Soc London 1852; 142: 463–562.
3. Coons AH, Creech HJ, Norman Jones R, Berliner E. The Demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J Immunol 1942; 45: 159–170.
4. Shimomura O, Johnson FH, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol. 1962;59:223-239.
5. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992;111(2):229-233.
6. Heim R, Prasher DC, Tsien RY. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA. 1994;91(26):12501-12504.
7. Terskikh A, Fradkov A, Ermakova G, et al. “Fluorescent timer”: protein that changes color with time. Science. 2000;290(5496):1585-1588.