## Commercial microscope stage incubators

1. Tokaihit
WKSM
silicone gas supply tube:
from facility to gas mixer: ODxID 10mmx6mm
from gas mixer to incubator: ODXID 6mmx4mm after one way valve ID2.5mm

## Building enforced humidifier for microscope stage incubators.

### How relative humidity (RH) affect on medium evaporation

at OIST, CO2 gas came from central gas cylinder than goes through tubings throughout the facility and the room RH is about 50%
The gas mixer from Tokai hit mixes CO2 gas with room air into 5% CO2. Tokai hit systems don't come with a humidifier.  The humidity into the chamber without water bath is about 20%.
if we add water into the water bath the humidity when stable can reach 75%.
However this is still low because in conventional incubator the RH is 95%.

I have the idea of building a humidifier using HPLC reservoirs. (from humidified respiratory mask in respiratory medicine, basically, the dry gas goes through a water reservoir to bring up the humidity to avoid discomfort to patients from drying)

Evidently, other incubator companies such as Pecon also have similar system to increase humidity. (such as one on Zeiss LSM880 confocal microscope system)

So to consider how relative humidity affects on medium evaporation:

Adren Buck equation (1981)
New equations for computing vapor pressure and enhancement factor, J Appl. Meterology, 20:1527-1552
https://en.wikipedia.org/wiki/Arden_Buck_equation

at 37°C the saturated vapor pressure for water is 6279.88Pa.
HUmidity ratio of moist air by vapor partial pressure can be calculated
=>0.0411Kg/Kg for 100% moist atmosphere.

as Relative humidity if defined as the ratio of partial pressure of water vapor over equilibrium vapor pressure.

RH75%
partial vapor pressure:4709.9Pa
humidity ratio=0.0303 Kg/Kg

RH90%
partial vapor pressure:5651.89Pa
humidity ratio=0.03698Kg/Kg

RH95%
partial vapor pressure:5965.9Pa
humidity ratio=0.0389 Kg/Kg

On e can further calculate evaporation rate
http://www.engineeringtoolbox.com/evaporation-water-surface-d_690.html
(Note:it's very rough)

75%:
g_s=9.99mg/hr
90%
g_s=3.811mg/hr
95%
g_s=2.035mg/hr

If in a microfluidic chip with two radius 2mm opening with height of 5mm and a strain channel of 8.8mmx1mmx0.1mm channel.
The total volume of medium is 126.5ul=126.5mg of water.
opening surface area is about 25.1 mm^2

the volume change of water in the microfluidic chip in different relative humidities can be calculated:
volume change = (126.5-(g_s*8))/126.5*100%
For 8 hrs.
RH75%
Δ=63.2%
RH90%
Δ=24.11%
RH95%
Δ=12.9%

Because the overall volume in a microfluidic chip is significantly less than that in a dish. And also when you use a dish, you can easily cover a lid to further prevent evaporation but it's difficult to cover a microfluidic chip.
The evaporation will can significant volume change in media that means the osmolarity can change also drastically. (water evaporates but amino acids etc in the medium won't)
The cells then are subject to significant metabolic stress and will result in microenvironmentally differences in comparison to standard incubator. (This will not come out nice)
Thus, to rectify humidity problem is essential.

We build a enforced humidifier to correct the RH problem in Tokai hit incubators and can successfully maintain RH to 90% without condensation on the chip obstructing the viewfield.

In addition, we can use silicone oil to cover the open surface to further avoid the evaporation problem.
But if the RH is not compensated, we still observe drying over time even with the silicone oil.

We can show the calculated results of evaporation rate and medium volume change for the above mentioned microfluidic channel under different relative humidities.

### Enforced Humidifier (V1)

Build humidifier using HPLC accessories

GL38 50mL bottle
cap:
OM4412

from gas mixer
ID2.5mm->VRM406->P-658->P-200x->XP-335x->1/8" tube into bottle under water

to chamber:
VRSR206->ID2.5mm

### Enforced Humidifier (V2)

GL45 1000mL bottle

cap:
OM4212 ￥2800
equivalent to Omnifit 00945T-2

from gas mixer:
ID2.5mm->PVDF 0.22um filter->VRM206->ID2.5mm->VRM206->P-658->P-200x->XP335X->1/8" FEP tubing->a PTFE filter (OM4244)

to chamber:
VRSR206->ID2.5mm

cap:2*OM4251S (no cap is needed if you're using OM4212 and not OM4214)

Humidifier based on surface acoustic wave nebulizer
https://product.tdk.com/info/ja/catalog/datasheets/nebulizer_nb_ja.pdf
http://www.seiko-giken.jp/items/2011/08/nb59s-n.php
https://www.honda-el.co.jp/product/hm2412.html

## How to make transparent planar temperature heater for cell culture

### Transparent planar heater using ITO glass or FTO coated glass

A conductive thin layer coated glass can be used as a transparent heater when a current passes through it.
However, it is necessary to apply a layer of "bus electrode" to facilitate electrical current propagation uniformly through the entire sheet of ITO.
We typically apply a layer of highly conductive silver ink with a thin slice of stainless steel from stainless steel gauge.

Comments