Creating a microscope page

A guide to using the microscope page creation tool on FPbase

TL;DR

If you already know whats on your microscope and you just want to get up and running quickly, format your information as follows and use the bulk-entry tab:

Green, ET470/40x, T495lpxr, ET525/50m
Red, FF01-543/22, FF562-Di03, FF01-593/40

Gather information

The first step in creating your customized spectra-viewer ("microscope page") is to gather the information about your instrument that you will need. This includes:

  • Light source(s), such as an LED or metal-halide light engine, or laser wavelengths

  • Excitation, dichroic (beamsplitters) and emission filters. You will need the actual brand and model number (for Chroma and Semrock) filters, or you will need to have the spectrum information available.

  • Camera(s)

If you don't know exactly what fluorescence filters you have in your instrument, then you've got to do some digging. Most mounted commercial filters will have the necessary information printed on the side of the filter, though dichroic filters are often unlabeled.

If you are not the person who maintains your microscope, and if you do not know exactly what you're doing, please ask for assistance before opening up your microscope!

In a conventional microscope, your filters will often be in a filter cube inside the filter turret. Alternatively, your excitation filters and/or emission filters may be mounted in external filter wheels.

filter cube image from Chroma.com

If you must open the cube or the wheels to see which filters you have, make sure to take note of the filter orientation, and replace the filters in the same orientation. Also, wear gloves to avoid getting finger prints on any of the optical surfaces.

Chroma part numbers will often be named something like ET525/50x, whereas Semrock part numbers typically look something like FF03-525/50. Though in each case, there are many variations.

Optical configurations

Next, you need to assemble a list of the different optical configurations that you use to image different fluorophores on your microscope. By "optical configuration" here, we are referring to a combination of light source, filters, and camera (these usually correspond to buttons or settings in your microscope software that put the appropriate hardware into the optical path). Here's an example of how such a table might look:

Config Name

Light Source

Ex Filter

Dichroic

Em Filter

Green

SOLA 395

ET470/40x

T495lpxr

ET525/50m

Red

SOLA 395

FF01-543/22

FF562-Di03

FF01-593/40

It's possible that a single filter (particularly multi-band dichroic filters) will be used in more than one optical configuration on your microscope.

Enter information at FPbase

You can create a new microscope at FPbase at https://www.fpbase.org/microscope/create/

After providing a name and (optional) description for your microscope, there are two ways to enter optical configuration information:

  1. In Bulk: This lets you enter everything as text, which can be much faster, particularly for a first-time setup, but it is more error prone, and requires that all the filters are already in the database. It's worth a try, then fall back to the manual form entry if necessary.

  2. One config at a time: Here, you select filters in each optical configuration in corresponding selection fields in the form. Slower, but less error prone.

Add Configs in Bulk

I recommend starting here, and resorting to the manual entry if you run into errors that cannot be easily fixed. To, add configs in bulk, select the middle tab

The bulk optical config tab on the microscope form

Bulk configs must be entered in the correct format: one config per line on a separate line, using the following 4 or 5 field comma-separated format:

Name, Laser OR Ex Filter(s), Dichroic(s), Em Filter(s) [, Dichcroic reflects ex?]

Commas are considered delimiters, and are not allowed in names. Lasers must be entered as integer values. Any broadband light sources entered in the "Light Source(s)" field, or cameras entered in the "Detector(s)" field will be applied to all of the configs, but leave them out of the text entry. (If you don't want the light source on all configs, you will have to add it manually later). For example, the two configs shown in the table above would be entered as follows:

Green, ET470/40x, T495lpxr, ET525/50m
Red, FF01-543/22, FF562-Di03, FF01-593/40

"Dichroics" here are implied to be in both the excitation and emission paths. If you have a dichroic that is only in the emission path, such as a beamsplitter used for simultaneous dual-color imaging, place that dichroic in the emission path (and, optionally, invert it... see below)

Inverted dichroic (e.g. Yokogawa orientation)

Most microscopes have a dichroic that is oriented such that it reflects excitation light to the sample, and passes emission light to the detector. Others (most commonly seen on a Yokogawa spinning disc confocal), have the dichroic in the opposite "inverted" arrangement, such that it reflects the emission light. An optional 5th item ("true" or "false") specifies whether the dichroic reflects excitation light. For example, a Yokogawa setup might look like this:

Yokogawa 488nm, 488, Di01-T405/488/568/647, ET525/50m, false

Multiple filters of a single type

In some cases, you might have multiple sequential filters in the same "role" (excitation or emission). For instance, some light sources have excitation filters inside of the light source, and you may also have a filter elsewhere in your light path. Alternatively, image splitting devices sometimes have a dichroic in the emission path as well as a cleanup-barrier filter. Multiple filters in a given part of the light path can be indicated by enclosing them in parentheses. For example, a TIRF scope that has a notch filter in addition to a barrier filter in the emission path:

TIRF 647nm, 647, ZT405/488/561/647rpc, (ZET405/488/561/647m, ET700/75m)

If you need to "invert" a filter or beamsplitter in the emission path, you will have to do that in the manual optical config setup after you save the form.

Edit Configs Manually

This is the default method (first tab), and allows finer-tuned control over each optical config, and is a bit less error-prone than the bulk method. It is pretty self-explanatory: just enter the appropriate filter, or laser/light source/camera in the appropriate field in the optical config. Multiple filters can be entered in each of the fields. If a filter does not exist, you can try to import it with the "import commercial filter" button, otherwise you will have to upload it manually. To add additional configs, click the "Add Optical Config" button.

Individual optical config forms

Inverted dichroics

As described above, some dichroic filters are arranged such that they reflect (rather than transmit) emission light. If that is the case (such as on most Yokogawa systems), click the "reflects em" box above the dichroic filters.

Reflective Emission Filters

Often in simultaneous dual-channel (image-splitting) setups, there is a (usually longpass) dichroic filter in the emission path that reflects shorter wavelengths. In that case, you may add the dichroic filter to the "Advanced: Reflective Emission Filter(s)" field (e.g. for the shorter wavelength in a simultaneous dual-channel setup).

Add Extra Components

The third tab allows you to enter additional components that you would like to have available, but which you don't want to have associated with any particular optical config