In biomedical laboratories, flow cytometry laser can analyze dozens of characteristics of individual cells simultaneously at a rate of tens of thousands of cells per second, covering everything from the identifying markers of immune cells to the special functions of stem cells and intracellular signal fluctuations. What gives this screening tool its precise detection capabilities is the “laser team” behind it. Different wavelengths of laser light act like different bands of illumination, precisely lighting up corresponding fluorescent dyes, leaving no secret of the cell hidden.

Figure: With the development of bright violet (BV) polymer dyes by BD Sirigen, violet laser diodes have become an important excitation source. Currently, there are several BV dyes (BV421, BV480, BV570, BV605, BV650, and BV787), which are spectrally compatible and significantly increase the number of analyzable simultaneous cell markers.

Figure: Dual immunofluorescence of hepatitis cells using Brilliant Violet 421 and 480 combined with secondary antibodies. Nuclear spots were stained with DRAQ5.

Four key members: core members with distinct roles.

The current laser team has four recognized core members, each undertaking a key function, and none of them can be missing.

Cyan 488nm: The All-Round Captain

This is the standard light source for all flow cytometers. It is responsible for measuring cell size (forward scattering) and internal complexity (side scattering), while efficiently exciting FITC, green fluorescent protein (GFP), phycoerythrin (PE), and their tandem dyes. These dyes are fundamental reagents for immunophenotyping and fluorescent protein research; therefore, the performance of 488nm directly determines the instrument’s basic detection capabilities. Modern 488nm light sources mostly use direct semiconductor lasers.

Red 633-640nm: The Main Source of Immunoassay.

It excites red fluorescent dyes such as APC and Cy5, whose emission wavelengths are far from the cell’s autofluorescence region, resulting in a high signal-to-noise ratio. Red laser diodes are inexpensive and compact, making them a standard feature in mid-to-high-end flow cytometers. Because the laser wavelength is quite close to the dye emission peak, a 640/8nm narrowband cleaning filter must be added to the laser front end to remove stray light, and a high-blocking emission filter must be used to prevent laser leakage into the detector.

Purple 405nm: A Pioneer in High-Dimensionality Flow Cytometry.

A key light source for high-dimensional flow cytometry analysis. Currently, over ten BV series dyes (BV421 to BV785) can be efficiently excited by it. These dyes have high brightness and low spectral overlap, significantly improving detection parameters. Furthermore, it can also excite traditional blue dyes such as Pacific Blue and Cascade Blue, as well as some quantum dot probes. Purple laser diodes are typically equipped with 405/10nm clean filters, with power ranging from 20mW to 200mW. 50-100mW power is sufficient for most applications (this refers to conventional analytical flow cytometers; sorters usually require higher power).

Green-Yellow 532-561nm: Fluorescent Protein Experts.

Used to optimize the excitation efficiency of PE and red fluorescent proteins. The maximum excitation peak of PE is at 554nm, and the excitation peaks of red fluorescent proteins such as DsRed and mCherry are also between 550-590nm. 488nm lasers have low excitation efficiency for these dyes, while 532nm green and 561nm yellow lasers can improve the excitation efficiency by more than 3 times. Simultaneously, using independent green-yellow lasers to excite PE can significantly reduce the spectral overlap with FITC, reducing the compensation value from approximately 16% to near 0%.

Ultraviolet (UV) lasers: The core component of high-end detection.

UV lasers were once a high-end feature of flow cytometers, equipped only in a few high-end instruments. With the development of high-dimensional flow cytometry and stem cell research, UV lasers have become an essential component of high-end detection.

375nm: A Cost-Effective Choice.

Developed to lower the barrier to entry for UV applications, this near-ultraviolet laser diode costs only a fraction of a 355nm DPSS laser. Its compact size allows for effective excitation of most BUV and Hoechst dyes, making it suitable for routine applications that do not require indo-1 detection. When using a 375nm laser to excite BUV395, a 386/23nm emission filter is required instead of the conventional 390/18nm filter to avoid interference from stray laser light. However, it has a significant drawback: it cannot excite indo-1, the gold standard probe for calcium signal detection, because the excitation peak of indo-1 is between 330-345nm, and the 375nm wavelength is too long, resulting in almost no effective excitation.

Future Outlook: Smaller, Stronger, More Diverse

Advances in laser technology have never ceased. The future of laser technology will develop in three directions: smaller size, driving the miniaturization and portability of high-end flow cytometers; higher power, improving the sensitivity of weak signal detection and enabling precise quantification of low-abundance proteins on cell surfaces; and more wavelengths, filling spectral gaps and further increasing the number of parameters that can be detected simultaneously.

BU-Laser recommends the following semiconductor lasers for flow cytometry:

1. Single Mode 4um Fiber 638nm 50mW Red Fiber Coupled Laser Module for Myringitis Treatment
2. 488nm 60mW Single-mode Fiber Laser Module for Biological Cell Imaging
3. 520nm 35mW Green Fiber Coupled Laser Module with 9um Fiber for Ophthalmological Treatment
4. 450nm 70mW Blue Fiber Coupled Laser Module for Medical Treatment
5. UV 375nm 200mW Fiber-coupled Laser Module