mCherry is a very popular red fluorescent protein. However it has several disadvantages:
- It is shifted towards far red (ex peak 585 nm) so it often is not imaged optimally with the illumination sources and filters commonly available.
- It bleaches fast
mCherry has now been evolved into mCherry-XL with several improvements:
- This variant is shifted back towards green (ex peak 560 nm) therefore being very well excited with popular 561nm lasers.
- It is 3 times brighter than mCherry
- There is also a clear improvement in the lifetime for FLIM
- Together the 2 points above means that less excitation power is required so it should help with the bleaching problem
Here is the paper. Therefore you should consider mCherry-XL for your future tagging with red fluorescence proteins.
It is now time to apply to the intensive LCI microscopy course Jan/Feb 2022: Microscopy: improve your imaging skills – from sample preparation to image analysis
The aim for this course is to improve the microscopy skills of students and researchers who have already used a microscope to acquire digital images of fluorescent samples but feel that their knowledge is limited.
All the lectures at the LCI microscopy course will also broadcasted live online, free of charge and there is no need to register.
All details about the course including course schedule, how to apply, and how to follow the lectures are found here.
Scroll down to read the kind testimonies of our dear students! 😊
Hope you enjoy the LCI facility microscopy course 2022!
Fluorophores are in constant development.
There is now a new generation of Alexa Fluor fluorophores: the Alexa Fluor Plus. They can be purchased already coupled to secondary antibodies on the ThermoFisher website.
Brilliant violet antibodies are also very bright and despite their name, they come in all colours. The can be purchased from Biolegend which produces the fluorophore.
Another excellent source of bright fluorophores is the Janelia Fluor dyes. They can be purchased coupled to antibodies from the NovusBio website.
As usual the lectures at the LCI microscopy course will broadcasted live online, free of charge and there is no need to register.
Title: Microscopy: improve your imaging skills – from sample preparation to image analysis
The aim for this course is to improve the microscopy skills of students and researchers who have already used a microscope to acquire digital images of fluorescent samples but feel that more knowledge could help them.
Applications are closed but all lectures will be broadcasted live and open to anyone without registration.
The course covers the following topics:
- Optics, image formation, fluorescence, fluorophores, microscope and microscopy types
- Objectives and refraction index, Cameras and detectors
- Noise and background, Cameras and detectors, Bit depth and saturation, Multicolour imaging
- Resolution and contrast, Sample preparation, Immunostaining
- Nyquist sampling, Confocal and wide field settings, Scaling up and speeding up, High throughput/content
- Volume imaging, deconvolution, multiphoton, Clearing and expansion
- Live cell imaging, Fourier, AI, Super Resolution microscopy
- Data handling, OMERO.figure, Requirements for image analysis, Colocalization
- Image processing and analysis
Check the course schedule and details of how to join the Zoom webinars. Scroll down to read the kind testimonies of our dear students! 😊
Here is the course syllabus.
Hope you enjoy the LCI facility microscopy course 2021!
Fluorophores are evolving fast! Here is a paper about a bunch of new fluorophores isolated from the jelly fish Aequoria. This includes the brightest fluorophore ever isolated: a new green fluorescent protein called AausFP1 that is almost 5 times brigther than GFP! Respect! 🙂
Do you know that clearing is not just about light sheet microscopy? Even if you have done your job well and your sample is directly on the coverslip (not on the slide), as soon as your sample is thicker than 10 um (1 cell diameter), you will see the effect of the refraction index mismatch.
What is that? Your sample and the mounting medium around it have a certain refraction index (or likely several). The objective you are using is designed for a certain refraction index (e.g. air, water or oil). If these refraction indices do not match what happens? as soon as you image a tiny bit away from the coverslip, the sample will look elongated, the intensity and contrast will drop very fast.
Sounds familiar? If yes, changing your mounting medium to match the objective will solve the problem. It works for light sheet but it also works for wide field or confocal imaging! Just change your mounting medium and you will see an enormous difference!
Here is an article describing a one-step clearing protocol. This basically is about using a different mounting medium. Easy, cheap and non-toxic! Give it a try!
Have a look at this post for more info.
Here you can see a nice film of a beating cardiomyocyte.
It was transfected using Fuse-it vesicles full of the mRNA of LifeAct-tagGFP2. According to Ibidi, it also work well with primary cells which are typically difficult to transfect.
RNA-based transfection seems to be gaining speed compared to classical transfections using DNA.
If you try it, leave some comments here to tell us how it worked for you! 🙂
Sounds good! No more buffer that stops working after one hour of imaging!
This company sells SaraFluor650 secondary antibodies. This fluorophore seems to be a natural blinker which does not need to reducing environment. Apparently it doesn’t need very bright lasers either!
Apparently they have also developed a green variant. So you can run 2-colour direct STORM on your favorite TIRF system! 😀
They also sell pH sensors and more so check their website and if you try, write a comment to let us know how things worked!
The LCI will host a seminar about GlyCLICK, a new way to directly label primary antibodies and stop using secondaries: 6th of November at 13:00 in the Lipid seminar room in Neo, KI Flemingsberg. The seminar will be broadcasted live. Here is how to find us and here how to follow the seminar online.
Everyone agrees that it would be great to be able to label our samples without using secondary antibodies.
- less animals killed
- shorter and cheaper protocols
- no problem with isotype cross-reaction
- no problem with secondary species when using many antibodies at once
There are many kits to label primary antibodies directly with fluorophores. The main disadvantage compared to primary/secondary stainings is that direct labels are often weaker because the final primary/fluorophore ratio is too low. Using an amplification method like TSA (Tyramide Signal Amplification) leads to a loss of resolution.
Over the years users at the LCI have tried this kit, this kit and this one. They gave mixed results depending on the antibody but we keep looking! Come to the LCI seminar about a new direct labelling technique that uses Click chemistry.
Measuring the volume of a cell is often done by labelling the cell membrane or its cytoplasm. Analysing large flat cells this way is easy but it is much harder for tiny cells like blood cells, yeast or bacteria.
Another way to measure volumes is to use a negative stain, i.e. where the medium is made fluorescent with a dye that does not go into the cell. The cell appears as a black hole in fluorescent images and unlike lipid-based membrane labelling, borders are even and easy to segment.
While many dyes can be used for live cells, one must choose large dyes when negatively imaging cells that have been fixed and permeabilized.
This paper and this one use high molecular weight (2000 KDa) Dextran to achieve these results and measure the size of bacteria.
This recent paper optimizes the technique.