The LCI open microscopy course starts on Monday! :)

Dear all

It is my pleasure to invite you to follow the LCI facility microscopy course Microscopy: improve your imaging skills – from sample preparation to image analysis.

The course starts next Monday (29 Jan) and runs until the 16th of February.

As usual, all the course lectures are broadcasted live on Zoom. It is free of charge and you do not need to register.

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.

Here is a selection of what we will talk about:

  • Optics and image formation,
  • Anatomy of a microscope
  • Objectives and refraction index
  • Cameras and detectors, Noise and background, Bit depth and saturation
  • Sample preparation, Immunostaining, Clearing and expansion
  • Resolution and contrast, Nyquist sampling, Microscope settings
  • Data handling, OMERO.figure, Requirements for image analysis, Colocalization
  • Image processing and analysis

Check our course webpage to see the course schedule (Broadcasted activities are in blue) and the Zoom link. Scroll down to read the kind testimonies of our dear students! πŸ™‚

Here is the course syllabus.

For those who are on a far away time zome, we record all the live lectures and post them every evening on the LCI facility YouTube channel! πŸ™‚

We hope that you will enjoy the LCI facility microscopy course!

Kindly forward to anyone who might be interested.

The LCI team

A few spots left at the LCI microscopy course 29/01-16/02 2024

There are a few spots left for the LCI core facility light microscopy course (29 Jan – 17 Feb 2024): β€˜Microscopy: improve your imaging skills – from sample preparation to image analysis’ (6 credits).

This course is completely unique in that it is a highly hands-on, but your hands will be on your own microscope and own sample. The course runs completely remotely! 😊

  • To see how the course can help your microscopy project, check the course webpage. Look at the course schedule 2024 and the alumni testimonies!
  • Also check the course syllabus to see the eligibility criteria. Also read what you will learn in the Course content and Intended Learning Outcomes sections.
  • If you cannot apply to the course, you can anyway follow any of the lectures (in blue on the schedule) as they will be publicly broadcasted live on Zoom and accessible to anyone without registration. The schedule and zoom link are available on the course page.

The purpose of the LCI facility microscopy course is to provide PhD students, researchers and core facility staff who have some prior experience of microscopy with enough theoretical and practical knowledge about their OWN sample and their OWN microscope, to enable them to:

1) assess and improve their sample so that it becomes suitable for data extraction from fluorescence images,

2) make best use of the hardware available in their lab/facility,

3) fully understand the acquisition parameters they need to set in their own microscope software,

4) design their experiment from scientific question to image analysis using a strong knowledge base.

The aim is to provide you with tools to acquire on ANY wide field, confocal or light sheet microscope, images of your samples that reliably answer your scientific question.

The course is free of charge. Contact us (LiveCellImaging@ki.se) for enquiries.

mCherry-XL: brigther, more stable and with better spectra!

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.

The LCI microscopy course 2022 is now open for registrations!

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!

Brighter secondary antibodies

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.

The LCI microscopy course 2021 starts next week! :)

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!

Super bright green fluorescent protein

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! πŸ™‚

One-step non-toxic clearing protocol

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.

Transfecting hard-to-transfect cells

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! πŸ™‚

Fluorophores that blink for STORM without any special buffer

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! πŸ˜€

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!

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