Brilliant Microscopy

 

Where BD Horizon™ V450, Pacific Blue™ and eFluor® 405 are just too dim for fluorescence microscopy, Brilliant Violet 421™ now enables visualization of antigens with directly conjugated antibodies, enhancing your capabilities with multi-color microscopy.


Historically, the "blue" channel on a microscope is reserved for an Hoechst or DAPI counterstaining due to the dim signal of typical dyes in that range, such as Alexa Fluor® 350, coumarin and Pacific Blue™ and the high sample autofluorescence often seen at that short wavelength of excitation. The following figures demonstrate that even on antibodies against markers that are relatively low in abundance, the Brilliant Violet 421™ direct conjugates can be used in imaging applications with high sensitivity. Further, they demonstrate extremely good photostability for repeated imaging and confocal z-stack capture. Also, these direct conjugates of Brilliant Violet 421™ demonstrate that there is no need for secondary antibodies or other amplificatiton techniques in order to achieve sufficient signal. Brilliant Violet 421™ will enable the addition of another color to a multicolor microscopy application on instruments capable of exciting this fluorophore between 360-420 nm.

Brilliant Stability

Brilliant Stability image

Figure A. Photostability Curves plotting Brilliant Violet 421™ with and without antifade in the mounting medium against Pacific Blue™ mounted with no antifade. Photobleaching was conducted on a 3i spinning disk confocal, laser power at 100%, 300ms exposures every 1 s for a duration of 130 seconds. The curve is plotted as percent fluorescence relative to the initial fluorescence intensity. Since data is normalized to 100%, it is not a reflection on the initial fluorescence intensity for each fluorophore as BV421™ is much brighter than Pacific Blue™, as demonstrated in Figure B. With or without antifade, BV421™ retains more than half-maximal signal even after 130 seconds. Pacific Blue™, on the other hand, loses fluorescence more quickly, showing only 50% intensity by 60 seconds. Prolong Gold was able to attenuate the loss of fluorescence intensity for BV421™ upon initial exposure to laser, particularly noticeable within the first 10 to 30 seconds. Since most images are typically acquired within that initial few seconds, it is recommended to use a mounting medium containing antifade to preserve the brightest signal.

 

Figure B. Still images were captured at 0, 10, 20, 60, and 120 seconds. Samples were human PBMCs labeled with Brilliant Violet 421™ CD3 or Pacific Blue™ CD45. CD45 was required for detection of Pacific Blue™ because of its higher abundance than CD3 on PBMCs. Even though Pacific Blue™ endured 60 seconds of exposure before it lost 50% of its intensity, it was no longer bright enough at 20 seconds to be useful in imaging. The Brilliant Violet™ conjugates, on the other hand, were extremely bright and stable with antifade. Even at 120 seconds, a useful image could still be captured when antifade was used.

Brilliant Imaging

For multi-color imaging, the use of directly conjugated antibodies is essential. Brilliant Violet™ brings new capabilities to the violet laser, allowing for bright staining of weakly expressed markers.

brilliant imaging image

NK92 cells (human NK cell line) were fixed and stained with anti-CD56 BV421, anti-perforin FITC, and phalloidin AF568 imaged on an Olympus IX81 spinning disk confocal microscope on 100X objective, NA 1.45. Exposures: 488 = 1000 ms, 568 = 100 ms, BV421™ (450 nm) = 200 ms. Data provided by Emily Mace and Jordan Orange, University of Pennsylvania.

Brilliant Violet 421™ Filter Choice

As previously mentioned, BV421™ is used in the “blue” channel, which is typically occupied by DAPI or Alexa Fluor® 405. However, as there is no set BV421™ filter on the market yet and because a “DAPI filter” may not be ideal, filter choice based on their wavelengths (excitation, emission and dichroic filters) is particularly important.

For example, the filter set in the image below works well as the dichroic filter (red line) does not block any of the BV421™ emission. However, in filter setups where this dichroic is set further to the right, a large percentage of the photons emitted by BV421™ may not reach the camera. If your filter setup is suitable, BV421™ can provide you with a bright, photostable option for multicolor microscopy.

 

 

BioLegend has validated the following filter setups for BV421™ (Ex: Excitation, Em: Emission):

  • Ex: 379/34, Dichroic: 409 LP, Em: 425/26
  • Ex: 395/25, Dichroic: 405 LP, Em: 440/40
  • Ex: 350/50, Dichroic: 400 LP, Em: 460/50

 

Brilliant High Throughput

BV421™ is useful for high thoughput content confocal imaging using the violet laser, providing clear and reliable results.

brilliant high throughput

Primary human macrophages in 384-well plates were polarized to M1 or M2, then fixed and stained with Brilliant Violet 421™ CD38. Plate was read on the ImageXpress Ultra from Molecular Devices, and fluorescence signal was quantitated across 6 replicates. The mean fluorescence + SD is plotted. Data provided by unnamed collaborator.